Characterization Of New Isolates Research Articles

Differential diagnosis of human Entamoeba infections: Morphological and molecular characterization of new isolates in Argentina

Entamoeba infections occur worldwide, with higher frequency in countries of low socioeconomic status and poor public health. Since Entamoeba histolytica has long been recognized as the only pathogenic species, making a differential diagnosis of other morphologically identical Entamoeba is important. This study aimed to determine the prevalence of Entamoeba species in two populations from Argentina, make a differential diagnosis by PCR and characterize Entamoeba isolates at the SSU rRNA gene. A total of 493 serial fecal samples were obtained from individuals in the provinces of Buenos Aires (n=210) and Misiones (n=283). Samples were examined by conventional methods (formalin–ethyl acetate and Willis flotation) and specific PCRs to differentiate Entamoeba species. Entamoeba isolates were characterized by sequencing a fragment of the SSU rRNA gene. The overall prevalence of Entamoeba infection was 12.4%, being more prevalent in Buenos Aires than in Misiones (14.8% vs. 10.6%). A case of E. histolytica confirmed by PCR and sequence analysis was reported for the first time in Buenos Aires. Moreover, new genetic data on Entamoeba coli and Entamoeba dispar were recorded. The phylogenetic analysis revealed a congruence between morphological characteristics and SSU rRNA gene sequences. This study increases the amount of information on the distribution of these species in Argentina and the region of the Americas.

First Detection of GES-5-Producing Escherichia coli from Livestock—An Increasing Diversity of Carbapenemases Recognized from German Pig Production

Resistance to carbapenems due to carbapenemase-producing Enterobacteriaceae (CPE) is an increasing threat to human health worldwide. In recent years, CPE could be found only sporadically from livestock, but concern rose that livestock might become a reservoir for CPE. In 2019, the first GES carbapenemase-producing Escherichia coli from livestock was detected within the German national monitoring on antimicrobial resistance. The isolate was obtained from pig feces and was phenotypically resistant to meropenem and ertapenem. The isolate harbored three successive blaGES genes encoding for GES-1, GES-5 and GES-5B in an incomplete class-I integron on a 12 kb plasmid (pEC19-AB02908; Acc. No. MT955355). The strain further encoded for virulence-associated genes typical for uropathogenic E. coli, which might hint at an increased pathogenic potential. The isolate produced the third carbapenemase detected from German livestock. The finding underlines the importance CPE monitoring and detailed characterization of new isolates.

Biochemical characterization of Pochonia chlamydosporia Q30 as biocontrol agent and plant growth promoter

Pochonia chlamydosporia is a natural soil fungi, which in recent years has gained importance for its ability to nematodes biocontrol and its beneficial plant interaction. The biochemical characterization of new isolates is important to determine their potential as biocontrol agent and plant growth promoter. In the present work, through the use of specific culture media, it was found that Pochonia chlamydosporia Q30 possesses chitinolytic activity, which could involve in the degradation of the egg wall and the cuticule of the juvenile nematode stages, amilolytic and pectinolytic activiy related to root colonization as endophyte was also found. Finally, phosphate solubilization (242.6 ± 27.1 mg/L) and indoleacetic acid production (10.0 ± 1.2 mg/L) were detected as plant gowth promoting properties, which were later verified in watermelon seedlings, whose inoculation with 105 chlamydospores, increased the height and the stem diameter in a 13 and 6 %, respectively. In conclusion, P. chlamydosporia Q30 has potential to be tested as biocontrol agent for nematodes and plant growth promoter.

Application of unsupervised clustering algorithm and heat-map analysis for selection of lactic acid bacteria isolated from dairy samples based on desired probiotic properties

Phenotypic and genotypic characterization of candidate probiotic strains are indispensable steps to the nourishments utilization. Hence, in this study, sixty-three lactic acid bacteria (LAB) isolates of traditional dairy origin already distinguished using pyrosequence-based 16S-rRNA profiling were characterized for their probiotic properties. The unsupervised clustering algorithm and heat-map analysis efficiency for selection of candidate probiotic strains by oppressing their phenotypic characteristics were tested. Results demonstrated that YP9, YP8, TP15, and ShP1 isolates show highest acid and bile tolerance, and Cell surface hydrophobicity. Moreover, CP3, CP12, TP15, TP16, TP17, ShP1, YP8, YP9, and CuP3 showed the highest BSH activity. Interestingly, the inhibitory effects of YP8, ShP1, and YP9 against Yersinia, Listeria, and Yersinia, respectively, were confirmed. Clustering and heat-map analysis effectively selected and distinguished nine Lactobacillus isolates (YP8, YP9, CP3, TP15-17, ShP1, CP12, and CuP3) as candidate probiotic strains. This study opens a new avenue on the selection and characterization of new isolates of bacteria based on their probiotics properties for future application in functional foods.

Molecular characterization of new isolates of the entomopathogenic fungus Beauveria bassiana and their efficacy against the tobacco caterpillar, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)

Thirteen fungal isolates, having characteristics similar to Beauveria bassiana, were isolated from field soils of Punjab, India, on Beauveria-specific selective medium (BS medium). Molecular screening of the B. bassiana isolates, using PCR amplification with B. bassiana-specific primer and nucleotide sequence analysis of ITS region, confirmed only three isolates as B. bassiana (now named as BbR1, BbR2, and BbR3) along with MTCC 2028 reference isolate. Comparative RAPD-PCR amplification with ten different RAPD primers showed that all the three isolates were closely related. Genetic relatedness dendrogram developed, using RAPD-PCR data by UPGMA, measured the quantitative description of genetic diversity and showed that the standard isolate MTCC2028 was 58% similar to new isolates. B. bassiana isolates achieved their maximum growth during the optimum incubation period of 7 days. Significant mortality rates of the tobacco caterpillar, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), were recorded at all the tested concentrations of conidial suspensions (103 to 107 conidia ml−1) of B. bassiana. BbR2 was found to be the most potential local isolate, causing the maximum mortality (83.33%) of S. litura, with LC50 value of 935.663 conidia ml−1, followed by BbR3 (74.33%), BbR1 (72.22%), and BbM1 (67.12%) isolates.

Complete characterization of new isolates of Neptunomonas phycophila leads to emend its description and opens possibilities of biotechnological applications.

Five strains were isolated from gonad of Great scallop (Pecten maximus) broodstock in a Norwegian hatchery. The study of 16S rRNA gene sequences showed that these isolates belong to Neptunomonas phycophila, a bacterium originally isolated from a symbiont of the anemone Aiptasia tagetes from Puerto Rico. The gyrB and rpoB genes sequences confirmed the affiliation of the scallop isolates to this species. Phenotypic characterization was performed and some differences between the Norwegian isolates and the type strain of N. phycophila were detected, such as ranges of temperature, pH, and tolerance to salinity or the use of several substrates as sole carbon source which lead to an emended description of the species. The strain 3CM2.5 showed phosphatidylethanolamine and phosphatidylglycerol as the major polar lipids. The whole genomes of the scallop strain 3CM2.5 and type strain of the species CECT 8716T were obtained and the annotation of these genomes revealed the presence of genes involved in degradation of aromatic compounds in both strains. Results obtained not only widen the geographical and host ranges of N. phycophila, but also point out possible biotechnological applications for this bacterial species.

Quantifying the growth of chlamydia suis in cell culture using high-content microscopy.

The porcine pathogen Chlamydia suis is widespread in pig farming. Isolation of Chlamydia suis in cell culture is crucial for the generation and characterization of new isolates. However, isolation of Chlamydia suis strains from field samples is fastidious. Therefore, we exploited high-content microscopy to quantify the growth of Chlamydia suis strains in different cell lines. We found that the cell line yielding optimal propagation of Chlamydia suis differed among isolates, and we identified cell lines outperforming those routinely used for chlamydial isolation. We conclude that adaptation of the propagation procedure to the origin of the putative field isolate is highly recommended to improve the recovery rate.

Aeromonas salmonicida: updates on an old acquaintance.

Aeromonas salmonicida is the oldest known infectious agent to be linked to fish disease and constitutes a major bacterial pathogen of fish, in particular of salmonids. This bacterium can be found almost worldwide in both marine and freshwater environments and has been divided into several sub-species. In this review, we present the most recent developments concerning our understanding of this pathogen, including how the characterization of new isolates from non-salmonid hosts suggests a more nuanced picture of the importance of the so‑called 'atypical isolates'. We also describe the clinical presentation regarding the infection across several fish species and discuss what is known about the virulence of A. salmonicida and, in particular, the role that the type 3 secretion system might play in suppressing the immune response of its hosts. Finally, isolates have displayed varied levels of antibiotic resistance. Hence, we review a number of solutions that have been developed both to prevent outbreaks and to treat them once they occur, including the application of pre- and probiotic supplements.

Rapid Molecular Detection of Genetically Diverted Foot and Mouth Disease Virus Serotype O During the Outbreak of 2012 in Egypt

Increasing the international trade of animals and their products and the continuous changing of Foot and Mouth Disease Virus (FMDV) lead to the rise of disease introductions and create an urgent need for laboratory methods facilitating a swift and sensitive confirmation of suspect outbreaks and fast characterization of new isolates. As FMDV is extremely contagious and affects all cloven-hoofed animals, it provides a continuous burden and risk to the livestock industries of the developing world, in particular due to mortality in young animals, weight and milk loss, lameness and the trade restrictions necessary to control the disease. The control of FMD in Egypt requires an accurate analysis of the newly introduced viral strains and an analysis of their relationship with the current circulating strains and the routinely used vaccines. This study evaluates a recently developed rapid Reverse-Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) for the identification of FMDV in samples from suspect cases. Samples were collected from gum, tongue epithelium, vesicular fluid, buccal and gum swabs, as well as saliva of infected cattle, buffalo and sheep and examined using two RT-qPCR of routine and high-speed formats using IRES1 and 3D-OIE primers. In addition, partial VP1 sequencing of some selected isolates was carried out for phylogenetic analysis. The high-speed RT-qPCR allowed a reliable diagnosis of FMDV in less than half an hour and can be used as a fast and valuable method for the monitoring and controlling of the foot and mouth disease. A new variant genotype (FMD-O/Eg/Mans/14) was circulating in Egypt during the outbreak of 2012 showing 98.5% identity to the isolated strain in Sudan (SUD_6_2008).

Characterization of New Isolates of Apricot vein clearing-associated virus and of a New Prunus-Infecting Virus: Evidence for Recombination as a Driving Force in Betaflexiviridae Evolution.

Double stranded RNAs from Prunus samples gathered from various surveys were analyzed by a deep-sequencing approach. Contig annotations revealed the presence of a potential new viral species in an Azerbaijani almond tree (Prunus amygdalus) and its genome sequence was completed. Its genomic organization is similar to that of the recently described Apricot vein clearing associated virus (AVCaV) for which two new isolates were also characterized, in a similar fashion, from two Japanese plums (Prunus salicina) from a French germplasm collection. The amino acid identity values between the four proteins encoded by the genome of the new virus have identity levels with those of AVCaV which fall clearly outside the species demarcation criteria. The new virus should therefore be considered as a new species for which the name of Caucasus prunus virus (CPrV) has been proposed. Phylogenetic relationships and nucleotide comparisons suggested that together with AVCaV, CPrV could define a new genus (proposed name: Prunevirus) in the family Betaflexiviridae. A molecular test targeting both members of the new genus was developed, allowing the detection of additional AVCaV isolates, and therefore extending the known geographical distribution and the host range of AVCaV. Moreover, the phylogenetic trees reconstructed with the amino acid sequences of replicase, movement and coat proteins of representative Betaflexiviridae members suggest that Citrus leaf blotch virus (CLBV, type member of the genus Citrivirus) may have evolved from a recombination event involving a Prunevirus, further highlighting the importance of recombination as a driving force in Betaflexiviridae evolution. The sequences reported in the present manuscript have been deposited in the GenBank database under accession numbers KM507061-KM504070.

Characterization of monoclonal antibodies against foot-and-mouth disease virus serotype O and application in identification of antigenic variation in relation to vaccine strain selection.

BackgroundFoot-and-mouth disease (FMD) has severe implications for animal farming which leads to considerable financial losses because of its rapid spread, high morbidity and loss of productivity. For these reasons, the use of vaccine is often favoured to prevent and control FMD. Selection of the proper vaccine is extremely difficult because of the antigenic variation within FMDV serotypes. The aim of the current study was to produce a panel of mAbs and use it for the characterization of new isolates of FMDV serotype O.ResultsA panel of FMDV/O specific mAb was produced. The generated mAbs were then characterized using the peptide array and mAb resistant mutant selection. Seven out of the nine mAbs reacted with five known antigenic sites, thus the other two mAbs against non-neutralizing sites were identified. The mAbs were then evaluated by antigenic ELISA for the detection of forty-six FMDV serotype O isolates representing seven of ten known topotypes. Isolates ECU/4/10 and HKN/2/11 demonstrated the highest antigenic variation compared to the others. Furthermore, the panel of mAbs was used in vaccine matching by antigenic profiling ELISA with O1/Manisa as the reference strain. However, there was no correlation between vaccine matching by antigenic ELISA and the gold standard method, virus neutralisation test (VNT), for the forty-six FMDV/O isolates. Nine isolates had particularly poor correlation with the reference vaccine strain as revealed by the low r1 values in VNT. The amino acid sequences of the outer capsid proteins for these nine isolates were analyzed and compared with the vaccine strain O1/Manisa. The isolate ECU/4/10 displayed three unique amino acid substitutions around the antigenic sites 1, 3 and 4.ConclusionsThe panel of mAbs is useful to monitor the emergence of antigenically different strains and determination of relevant antigenic site differences. However, for vaccine matching VNT remains the preferred method but a combination of VNT, antigenic profiling with a panel of mAbs and genetic sequencing would probably be more ideal for full characterization of any new outbreak isolates as well as for selection of vaccine strains from FMDV antigen banks.

Characterization of a New Vaccinia virus Isolate Reveals the C23L Gene as a Putative Genetic Marker for Autochthonous Group 1 Brazilian Vaccinia virus

Since 1999, several Vaccinia virus (VACV) isolates, the etiological agents of bovine vaccinia (BV), have been frequently isolated and characterized with various biological and molecular methods. The results from these approaches have grouped these VACV isolates into two different clusters. This dichotomy has elicited debates surrounding the origin of the Brazilian VACV and its epidemiological significance. To ascertain vital information to settle these debates, we and other research groups have made efforts to identify molecular markers to discriminate VACV from other viruses of the genus Orthopoxvirus (OPV) and other VACV-BR groups. In this way, some genes have been identified as useful markers to discriminate between the VACV-BR groups. However, new markers are needed to infer ancestry and to correlate each sample or group with its unique epidemiological and biological features. The aims of this work were to characterize a new VACV isolate (VACV DMTV-2005) molecularly and biologically using conserved and non-conserved gene analyses for phylogenetic inference and to search for new genes that would elucidate the VACV-BR dichotomy. The VACV DMTV-2005 isolate reported in this study is biologically and phylogenetically clustered with other strains of Group 1 VACV-BR, the most prevalent VACV group that was isolated during the bovine vaccinia outbreaks in Brazil. Sequence analysis of C23L, the gene that encodes for the CC-chemokine-binding protein, revealed a ten-nucleotide deletion, which is a new Group 1 Brazilian VACV genetic marker. This deletion in the C23L open reading frame produces a premature stop-codon that is shared by all Group 1 VACV-BR strains and may also reflect the VACV-BR dichotomy; the deletion can also be considered to be a putative genetic marker for non-virulent Brazilian VACV isolates and may be used for the detection and molecular characterization of new isolates.

The use of two complementary DNA assays, AFLP and MLSA, for epidemic and phylogenetic studies of pectolytic enterobacterial strains with focus on the heterogeneous speciesPectobacterium carotovorum

Amplified fragment length polymorphism (AFLP) markers and multilocus sequence analysis (MLSA) were used to analyse 63 bacterial strains, including 30 soft‐rot‐causing bacterial strains collected from Syrian potato fields and 33 reference strains. For the MLSA, additional sequences of 41 strains ofPectobacteriumandDickeya, available from the NCBI GenBank, were included to produce a single alignment of the 104 taxa for the seven concatenated genes (acnA,gapA,proA,icd,mtlD,mdhandpgi). The results indicate the need for a revision of the previously classified strains, as some potato‐derivedPectobacterium carotovorumstrains were re‐identified asP. wasabiae. The strains that were classified asP. carotovorumduring the analyses demonstrated high heterogeneity and grouped into fiveP. carotovorumhighly supported clusters (PcI to PcV). The strains represented a wide range of host plants including potatoes, cabbage, avocados, arum lilies, sugar cane and more. Host specificity was detected in PcV, in which four of the six strains were isolated from monocotyledonous plants. The PcV strains formed a clearly distinct group in all the constructed phylogenetic trees. The number of strains phylogenetically classified as subspecies ‘P. c. subsp. brasiliensis’ in PcIV dramatically increased in size as a result of the characterization of new isolates or re‐identification of previousP. carotovorumandP. atrosepticumstrains. TheP. carotovorumstrains from Syria were grouped into PcI, PcII and PcIV. This grouping indicates a lack of correlation between the geographical origin and classification of these pathogens.

Phenotypic variability and phylogenetic relationships of the genera Tolypothrix and Calothrix (Nostocales, Cyanobacteria) from running water

The taxonomy of heterocystous cyanobacteria belonging to the genera Calothrix and Tolypothrix has long been a matter of debate, but their phylogenetic relationships are still not well understood. Our aim was to compare the phylogeny and morphology of members of these genera, which exhibit basal-apical polarity. A phylogeny was reconstructed on the basis of 16S rRNA gene sequences and compared with the morphological characterization of new isolates and environmental samples. Strains isolated from several rivers and streams showed a high degree of tapering when they were cultured in a nutrient-rich medium. However, clear differences were apparent when they were transferred to a nutrient-poor medium. Some strains showed a low degree of tapering and other morphological features corresponding to the genus Tolypothrix, such as false branching, whereas others maintained the morphological characteristics of the genus Calothrix. Phylogenetic analysis was congruent with the phenotypic characterization, in which the strains and environmental samples of the Tolypothrix and Calothrix morphotypes could be clearly separated. Isolates with a low degree of tapering and natural samples of Tolypothrix distorta were grouped in the same cluster, but strains of the genus Calothrix fell into well separated clades. Results from this study showed that representatives of the genus Tolypothrix share most morphological and developmental properties and a high degree of 16S rRNA gene sequence similarity. However, although similar and sometimes overlapping morphologies may occur in isolates of the genus Calothrix, these morphotypes may be distinguished on the basis of their clear genetic divergence.

Molecular and biological characterization of new isolates of Cydia pomonella granulovirus from Iran

The Cydia pomonella granulovirus (CpGV) is a very effective biological control agent of the codling moth, Cydia pomonella L. (Lep.: Tortricidae). Only a few CpGV isolates originating from Mexico (M), England (E), and Russia (R) have been described so far. In a field survey at different locations in Iran, CpGV isolates were collected from single or pooled codling moth larvae. The isolates, designated I1, I7, I8, I15, I22, I28, I30, I66, I67, I68, and I70 showed genetic (DNA restriction endonuclease profiles) and biological (bioassays) differences. Most isolates could be attributed to genome types similar to those found in CpGV-M, -E, and -R. Some of them were clear mixtures of different genotypes. Thus, the CpGV isolates found in the North–West of Iran make an important contribution to the known diversity of CpGV. The occurrence of novel, naturally occurring CpGV isolates emphasize the necessity of further studies towards the diversity and evolution of CpGV.

Avian infectious bronchitis: characterization of new isolates from Italy.

The isolation of four new variants or serotypes of avian infectious bronchitis virus in Italy is reported. The antigenic characteristics of these strains were investigated by cross-neutralization tests with the new isolates, Fa 6881/97, AZ 27/98, AZ 20/97, and BS 216/01; two of the most common European serotypes, AZ 23/74 and CR 88121 (793B); and the classic Massachusetts M41 serotype in association with a panel of 17 specific antisera. On the basis of the results obtained, the new isolates show relevant serologic differences. In fact, the four isolates were not neutralized by antisera against the most common European and American serotypes; the AZ 20/97 isolate was partially neutralized by FA 6881/97 antiserum but not reciprocally. The closely related Fa 6881/97 and AZ 27/98 isolates can be considered rather diffused in our country because they have been isolated over 20 times in the last 3 yr in different parts of Italy. On the contrary, the AZ 20/97 and BS 216/01 isolates were reported only once so far. The reverse transcription-polymerase chain reaction showed that Fa 6881/97 isolate is related to 793B isolate, whereas AZ 27/98 and BS 216/01 isolates appeared not to be related to the most common European and Massachusetts serotypes.

Isolation from turkey breeder hens of a reassortant H1N2 influenza virus with swine, human, and avian lineage genes.

Type A influenza viruses can infect a wide range of birds and mammals, but influenza in a particular species is usually considered to be species specific. However, infection of turkeys with swine H1N1 viruses has been documented on several occasions. This report documents the isolation of an H1N2 influenza virus from a turkey breeder flock with a sudden drop in egg production. Sequence analysis of the virus showed that it was a complex reassortant virus with a mix of swine-, human-, and avian-origin influenza genes. A swine influenza virus with a similar gene complement was recently reported from pigs in Indiana. Isolation and identification of the virus required the use of nonconventional diagnostic procedures. The virus was isolated in embryonated chicken eggs by the yolk sac route of inoculation rather than by the typical chorioallantoic sac route. Interpretation of hemagglutination-inhibition test results required the use of turkey rather than chicken red blood cells, and identification of the neuraminidase subtype required the use of alternative reference sera in the neuraminidase-inhibition test. This report provides additional evidence that influenza viruses can cross species and cause a disease outbreak, and diagnosticians must be aware that the variability of influenza viruses can complicate the isolation and characterization of new isolates.

Random amplified polymorphic DNA profiles as a tool for the characterization of Brazilian keratitis isolates of the genus Acanthamoeba.

The genus Acanthamoeba comprises free-living amebae identified as opportunistic pathogens of humans and other animal species. Morphological, biochemical and molecular approaches have shown wide genetic diversity within the genus. In an attempt to determine the genetic relatedness among isolates of Acanthamoeba we analyzed randomly amplified polymorphic DNA (RAPD) profiles of 11 Brazilian isolates from cases of human keratitis and 8 American type culture collection (ATCC) reference strains. We found that ATCC strains belonging to the same species present polymorphic RAPD profiles whereas strains of different species show very similar profiles. Although most Brazilian isolates could not be assigned with certainty to any of the reference species, they could be clustered according to pattern similarities. The results show that RAPD analysis is a useful tool for the rapid characterization of new isolates and the assessment of genetic relatedness of Acanthamoeba spp. A comparison between RAPD analyses and morphological characteristics of cyst stages is also discussed.

Cadmium binding by bacteria: screening and characterization of new isolates and mutants

A fast and simple methodology was developed that enables screening of microbial strains for their ability to bind cadmium. It is based on the use of a radioisotope of cadmium ( 109Cd) for screening colonies and for evaluation of cadmium binding. The methods described here can be used to screen new environmental isolates or to obtain mutants with altered ability to bind cadmium. Examples for the two uses are described in the paper.

Cadmium binding by bacteria: screening and characterization of new isolates and mutants.

A fast and simple methodology was developed that enables screening of microbial strains for their ability to bind cadmium. It is based on the use of a radioisotope of cadmium (109Cd) for screening colonies and for evaluation of cadmium binding. The methods described here can be used to screen new environmental isolates or to obtain mutants with altered ability to bind cadmium. Examples for the two uses are described in the paper.