Partial 16S rRNA Gene Sequences Research Articles

Achromobacter sp. Strain BUKˍBCHˍTQ1: A Potential Paraquat-Degrading Bacterium Isolated from Pesticide Contaminated Agricultural Soil

Paraquat (1, 1′-dimethyl-4, 4′-bipyridinium dichloride) is one of the most frequently used herbicide in agriculture. It is a cationic non-systematic, non-selective contact compound that instantaneously interferes with the photosynthetic processes of plants. It has an immediate effect, once the compound comes into contact with the plants’ leaves, where the reaction occurs. However, the contamination of paraquat residue in soil can harm soil microbes, flora and fauna, farmer health and also soil ecology, which affects the soil fertility. The objective of this study was to isolate and characterize bacteria with the ability to break down and utilize paraquat as the primary carbon source. The isolation process involved the enrichment of mineral salt media (MSM) using serial dilution. The isolated bacterium underwent morphological, biochemical, and molecular identification following characterization. Results showed the isolate was identified as Achromobacter sp. with the accession number OQ372943 based on partial 16S rRNA gene sequence and phylogenetic analysis. The growth and degradation of paraquat by this isolate were optimum at a pH of 6.5, 276 mgL-1 of the substrate (paraquat), temperature of 35 °C, 200 µL of biomass size, and 48 h of incubation. The degradation efficiency of the isolate after 120 hours of incubation under optimal conditions was 91.01%. Hence, these results demonstrated a very high efficiency of paraquat degradation. Consequently, this isolate holds significant promise for paraquat degradation and could serve as a viable contender for remediating paraquat-contaminated environments.

Agronomic strategies to enhance the early vigor and yield of maize. Part I: the role of seed applied biostimulant, hybrid and starter fertilization on rhizosphere bacteria profile and diversity.

The sustainable intensification of maize-based systems may reduce greenhouse-gas emissions and the excessive use of non-renewable inputs. Considering the key role that the microbiological fertility has on crop growth and resilience, it is worth of interest studying the role of cropping system on the rhizosphere bacterial communities, that affect soil health and biological soil fertility. In this work we monitored and characterized the diversity and composition of native rhizosphere bacterial communities during the early growth phases of two maize genotypes of different early vigor, using a nitrogen (N)-phosphorus (P) starter fertilization and a biostimulant seed treatment, in a growth chamber experiment, by polymerase chain reaction-denaturing gradient gel electrophoresis of partial 16S rRNA gene and amplicon sequencing. Cluster analyses showed that the biostimulant treatment affected the rhizosphere bacterial microbiota of the ordinary hybrid more than that of the early vigor, both at plant emergence and at the 5-leaf stage. Moreover, the diversity indices calculated from the community profiles, revealed significant effects of NP fertilization on richness and the estimated effective number of species (H2) in both maize genotypes, while the biostimulant had a positive effect on plant growth promoting community of the ordinary hybrid, both at the plant emergence and at the fifth leaf stage. Our data showed that maize genotype was the major factor shaping rhizosphere bacterial community composition suggesting that the root system of the two maize hybrids recruited a different microbiota. Moreover, for the first time, we identified at the species and genus level the predominant native bacteria associated with two maize hybrids differing for vigor. These results pave the way for further studies to be performed on the effects of cropping system and specific crop practices, considering also the application of biostimulants, on beneficial rhizosphere microorganisms.

Anaplasma phagocytophilum in European bison (Bison bonasus) and their ticks from Lithuania and Poland

The increasing population of European bison (Bison bonasus) can contribute to the prevalence of zoonotic pathogens. The aim of the present study was to assess the presence of A. phagocytophilum infection in European bison tissues as well as ticks removed from European bison in Lithuania and Poland. A further objective of this work was to compare the detected A. phagocytophilum strains. A total of 85 tissue samples (spleen) of European bison and 560 ticks belonging to two species, Ixodes ricinus (n = 408) and Dermacentor reticulatus (n = 152) were tested. DNA of A. phagocytophilum was detected based on RT-PCR in 40% of the European bison samples, 8.8% of the I. ricinus and 5.9% of the D. reticulatus ticks. Analysis of the obtained partial 16S rRNA gene sequences of A. phagocytophilum revealed the presence of three variants with two polymorphic sites. Furthermore, phylogenetic analysis with partial msp4 gene sequences grouped A. phagocytophilum variants into three clusters. This study revealed that the groEL gene sequences of A. phagocytophilum from European bison and their ticks grouped into ecotype I and only one sequence from Lithuanian European bison belonged to ecotype II. The results of the present study indicated that European bison may play a role as a natural reservoir of A. phagocytophilum.

Lactobacilli Profile in Faecal Samples of Tunisian Children Diagnosed with Autism Spectrum Disorder

Alterations in faecal lactobacilli in autistic children have been reported, but little is known related to age and disorder severity. We used a culture-based method and partial 16S rRNA gene sequencing to isolate and identify lactobacilli strains from faeces of Tunisian autistic children (ASD group) and compared them with strains isolated from siblings (SIB) and children from the general population (GP). The ASD group displayed an increased number of different species compared to SIB and GP. Differences in species abundance with age accounted for a significant decrease in the abundance of Lactiplantibacillus plantarum/Lactiplantibacillus pentosus isolates in the GP at the age of 8–10 years compared to the age of 4–7 years, and to a significantly lower abundance of Lacticaseibacillus rhamnosus in the ASD group with respect to SIB and the GP at the age of 8–10 years. Simpson’s and Shannon–Wiener indices showed a more pronounced species diversity increase with age in the GP group compared to the ASD and SIB groups. Minor differences were found in lactobacilli prevalence and in species diversity between children with severe and mild-to-moderate ASD. Overall, we found substantial differences in the profile of faecal lactobacilli species in the ASD and GP groups at the age of 8–10 years.

Distribution and molecular identification of ixodid ticks infesting cattle in Kilombero and Iringa Districts, Tanzania

BackgroundHard ticks infesting cattle are vectors of Tick-borne diseases that causes major public health problems and considerable socioeconomic losses to the livestock industry in tropical and subtropical countries. A repeated cross-sectional study was carried out by collecting ticks on cattle during the wet and dry seasons from January to August 2021 in order to determine hard tick prevalence, distribution, and abundance on cattle in Kilombero and Iringa Districts of Tanzania. The collected ticks were identified morphologically using published morphological keys under a stereomicroscope and confirmed by polymerase chain reaction (PCR) and sequencing.ResultsOut of 740 examined cattle, 304, (41.08%) were infested with ticks. In total, 1,780 ticks were counted on one side of the animal’s body and doubled, whereby resulting in a total of 3,560 ticks were recorded. Individual tick burden ranged from n = 2 to 82 ticks per animal, with a mean tick burden of n = 11.7 ± 0.68 per animal. A total of 1,889 ticks were collected from infected cattle including 109 additional ticks observed while collecting ticks based on the animal’s posture when restrained to the ground. Out of 1,889 ticks, nine species from three genera were identified morphologically: 1,377 fit in the genus Rhipicephalus, 459 to the genus of Amblyomma, and 53 to the genus Hyalomma. Rhipicephalus microplus was the most prevalent (n = 909, 48.1%), followed by Rhipicephalus evertsi (n = 310, 16.4%), Amblyomma lepidum (n = 310, 16.4%), Rhipicephalus appendiculatus (n = 140, 7.4%), Amblyomma gemma (n = 120, 6.4%), Hyalomma rufipes (n = 50, 2.6%), Amblyomma variegatum (n = 29, 1.5%), Rhipicephalus decoloratus (n = 18, 1.0%), while the least common was Hyalomma albiparmatum (n = 3, 0.2%). Tick diversity was higher in Iringa compared to Kilombero District. Tick prevalence was higher in wet season (n = 148, 41.11%). Ticks were widely distributed in different parts of the host body, with the highest distribution on zone 4 (n = 1,060, 56.11%), which includes groin, flank, abdomen, and around inner thigh of the hind legs and the lowest distribution on zone 2 (n = 14, 0.74%), which includes the back surface of the body. Both Amblyomma lepidum and Rhipicephalus microplus species were found in all the five body zones, and with the highest proportions recorded on zone 4: A. lepidum (n = 209, 67.42%) and R. microplus (n = 714, 78.55%). The nine tick species identified morphologically were also confirmed using molecular methods. However, during sequencing, two species (Rhipicephalus appendiculatus and R. decoloratus) had poor quality sequences and were excluded from the sequence analysis. Sequencing results indicated high nucleotide identity (96–100%) with sequences available in GenBank and Barcode of Life Database (BOLD). The phylogenetic analysis of partial mitochondrial COI and 16S rRNA gene sequences of ticks were used to confirm the morphological identification.ConclusionThe results showed a high burden of tick infestation on cattle, which could reduce animal production and potentially increase the risk of tick-borne diseases. Therefore, it is necessary to explore the epidemiological and molecular aspects of various tick species in other regions of Tanzania.

Paracholeplasma manati sp. nov. isolated from Florida manatees (Trichechus manatus latirostris).

Four novel strains of a member of the genus Paracholeplasma (OakleyT, Holly, Lorelei and Ariel) were isolated from skin of Florida manatees (Trichechus manatus latirostris). These strains were phenotypically and genetically characterized and compared with the known species of the genera Acholeplasma (A.), Alteracholeplasma (Al.), Haploplasma (H.), Paracholeplasma (P.) and Mariniplasma (M.). All the strains produced acid from glucose but did not hydrolyze arginine or urea. All were propagated in ambient air supplemented with 5±1 % CO2 at 35-37 °C using SP4-Z, Columbia and brain-heart infusion medium. Colonies on solid medium showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma-like cellular morphology. The results of phylogenetic analyses based on partial 16S rRNA, rpoB, gyrB and parE gene sequences and the whole proteome data indicated that the novel species is a unique species but phylogenetically closely related to Paracholeplasma vituli, Paracholeplasma morum and 'Paracholeplasma brassicae'. The average nucleotide identity and digital DNA-DNA hybridization values between strain OakleyT and the closely related species were significantly lower than the accepted thresholds for describing novel prokaryotic species at the genomic level. On the basis of the genomic, phenotypic and phylogenetic properties, the novel strains represent a novel species of the genus Paracholeplasma, for which the name Paracholeplasma manati sp. nov. with the type strain OakleyT (=NCTC 14352T =DSM 110686T) is proposed. The genomic DNA G+C content and complete draft genome size for the type strain are 38.35 % and 1 873 856 bp, respectively.

Bambara Nut Root-Nodules Bacteria from a Semi-Arid Region of South Africa and Their Plant Growth-Promoting Traits.

Rhizobial nitrogen-fixing bacteria are the main inhabitants of the root nodules of legume plants. Studying the bacterial community of legume nodules is important in understanding plant growth and nutrient requirements. Culture-based technique was used to examine the bacterial community of these underground organs from Vigna subterranea L. Verdc (Bambara nut), an underutilized legume in Africa, for plant growth-promoting traits. In this study, Bambara nuts were planted to trap root-nodule bacteria, and the bacteria were morphologically, biochemically, and molecularly characterized. Five selected isolates were screened in vitro for their plant growth-promoting traits and exhibited differences in their phenotypic traits. The polymerase chain reaction (PCR) products were subjected to partial 16S rRNA gene sequencing for phylogenetic analysis. Based on 16S rRNA gene sequence, the isolates were identified as BA1 (Stenotrophomonas maltophilia), BA2 (Chryseobacterium sp.), BA3 (Pseudomonas alcaligenes), BA4 (Pseudomonas plecoglossicida), and BA5 (Pseudomonas hibiscicola). Results showed that four of the five isolates could produce IAA. The capability to solubilize phosphate in Pikovskaya's agar plates was positively shown by four isolates (BA2, BA3, BA4, and BA5). Three isolates could produce hydrogen cyanide while isolates BA1, BA3, BA4, and BA5 were found to have ammonia-production traits. The results suggest that these plant growth-promoting isolates can be used as inoculants for plant growth and productivity.

Plasmid permissiveness of wastewater microbiomes can be predicted from 16S rRNA sequences by machine learning.

Wastewater treatment plants (WWTPs) harbor a dense and diverse microbial community. They constantly receive antimicrobial residues and resistant strains, and therefore provide conditions for horizontal gene transfer (HGT) of antimicrobial resistance (AMR) determinants. This facilitates the transmission of clinically important genes between, e.g. enteric and environmental bacteria, and vice versa. Despite the clinical importance, tools for predicting HGT remain underdeveloped. In this study, we examined to which extent water cycle microbial community composition, as inferred by partial 16S rRNA gene sequences, can predict plasmid permissiveness, i.e. the ability of cells to receive a plasmid through conjugation, based on data from standardized filter mating assays using fluorescent bio-reporter plasmids. We leveraged a range of machine learning models for predicting the permissiveness for each taxon in the community, representing the range of hosts a plasmid is able to transfer to, for three broad host-range resistance IncP plasmids (pKJK5, pB10, and RP4). Our results indicate that the predicted permissiveness from the best performing model (random forest) showed a moderate-to-strong average correlation of 0.49 for pB10 [95% confidence interval (CI): 0.44-0.55], 0.43 for pKJK5 (0.95% CI: 0.41-0.49), and 0.53 for RP4 (0.95% CI: 0.48-0.57) with the experimental permissiveness in the unseen test dataset. Predictive phylogenetic signals occurred despite the broad host-range nature of these plasmids. Our results provide a framework that contributes to the assessment of the risk of AMR pollution in wastewater systems. The predictive tool is available as an application at https://github.com/DaneshMoradigaravand/PlasmidPerm.

First report of association of 'Candidatus Phytoplasma asteris' with Moringa oleifera leaf yellowing and stunting disease in India.

Moringa oleifera (family Moringaceae) also known as the 'drumstick tree' is a significant nutritious and medicinal plant that is commonly grown in India and contains a variety of vital phytochemicals. M. oleifera is used in several Indian herbal medicine formulations to treat a variety of illnesses (Kumar and Rao 2021). Typical phytoplasma symptoms of leaf yellowing and stunting were observed in M. oleifera trees up to 10% incidence at Acharya Narendra Dev University of Agriculture & Technology, Ayodhya, Uttar Pradesh, India in November 2021 and stunting with less fruit bearings symptoms with 8% incidence in October 2021 at Jonnalakothapalle village of Mudigubba mandal of Ananthapuramu district in Andhra Pradesh, India (Fig.1a, b). To investigate the possibility of a phytoplasma association with the symptoms, total DNA was isolated from the leaf samples collected from two diseased and two healthy plants from both the locations using CTAB method. The DNAs isolated were analysed by nested polymerase chain reaction (PCR) with universal phytoplasma primer pairs P1/P7 and R16F2n/R16R2 for the 16S rRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and secAfor1/sArev3 and SecAfor2/ SecArev3 for secA gene (Hodgetts et al. 2008). Amplicons of the expected size (~1.25kb from 16S rRNA gene and ~480bp from secA gene) were obtained from symptomatic plants only. The nested PCR products were cloned (pGEM-T Easy Vector, Promega), sequenced (ABA Biotech, India) and the sequences were deposited in GenBank with accession numbers OP358449, OP358450, OP358451, OP358452 for the 16SrRNA gene (~1.25 kb) and OP358443, OP358444, OP358445, OP358446 for the secA gene (~480 bp). BLASTn analysis revealed that the partial 16S rRNA gene sequences of M. oleifera phytoplasma isolate shared up to 99.9% sequence identity with the strain 'Candidatus Phytoplasma asteris' (Accession numbers MN909051, MN909047) and secA gene sequences shared up to 100% sequence identity with 'Ca. Phytoplasma asteris' (Accession numbers KJ434315, KJ462009) belonging to 16SrI group. The 16S rRNA and secA genes sequence-based phylogenetic analysis (Figure 1d,e) showed that the phytoplasma strain associated with M. oleifera leaf yellowing and stunting clustered within the 16SrI phytoplasma group closest to 16SrI-B ('Ca. P. asteris') subgroup strains. Furthermore, the virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment is identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession: AP006628). To the best of our knowledge, this is the first report of the 16SrI-B subgroup of the phytoplasma strains with M. oleifera in the world. 'Candidatus Phytoplasma asteris' (16SrI-B subgroup) strains have been reported from several other commercial crops and weed hosts in India and efficient leafhopper vectors have been identified (Rao 2021; Reddy 2021). This indicates that the 'Ca. P. asteris'-related strains (16SrI-B) are widespread and infecting several plant species in India. The increasing incidence of the 16SrI-B strain and its wide host range in India strongly suggests further research into the epidemiology involved in the dynamic spread of the disease in order to recommend a suitable management approach.

Energy from Waste: Poterioochromonas malhamensis Used for Managing Dairy Effluent and Producing Valuable Microalgal Lipid

Currently, microalgae have become a marvelous and resource-friendly alternative source of advantageous bioproducts, such as lipids, carbohydrates, proteins, or other bioactive compounds. Because of the richness of microalgae in these high-value-added metabolites, still, it is an underdeveloped source of sustainable energy and food. There are some hurdles to profitable production, such as culture contamination and costly harvesting techniques. In the current work, a chrysophyte was isolated from dairy wastewater, identified as Poterioochromonas malhamensis based on its morphology and partial 18S rRNA gene sequences. This isolate was used to remediate dairy waste water (DWW) and to obtain neutral lipids (fatty acids) from microalgae. Microalgal growth was optimized by using different concentrations of DWW, supplemented with all the nutritive requirements for better progression and flourishment. Maximum biomass yield 1.478 g L−1 was achieved by optimized cultural conditions (different concentrations of DWW with BBM media). This strain showed high nitrate and phosphate removal efficiency (87.45% and 88.96%), respectively in 15 days. The experimental results highlighted that the lipid content and the chemical oxygen demand (COD) removal were 31.60% and 88.84%, respectively, and the lipid profile of isolated microalga was C16:0, C16:1, C18:0, C18:1, and C18:2 fatty acids. For growth and treatment purposes, 75% DWW with Bold’s Basal Medium (BBM) media showed better results. This is the first report of DWW treatment using the microalga Poterioochromonas malhamensis, as far as we are aware. Its cultivation prevented the spread of pollution of freshwater sources, remedied the DWW, and generated important lipids for industry.

A new small-celled naviculoid diatom species, Mayamaea pannonica sp. nov. (Bacillariophyceae) from soda pans in Serbia

ABSTRACT Combining morphological and molecular approaches, a new diatom species is described in the genus Mayamaea from a Special Nature Reserve, Okanj bara in Serbia, an alkaline and subsaline soda pan with pronounced seasonal drying. Mayamaea pannonica sp. nov. possesses typical characteristics of the genus Mayamaea, such as uniseriate striae and hymenes that cover the areolae from the outside. It can easily be distinguished from similar species by the valve outline and shape of the central area. Although presently no culture of the species is available, we identified in an amplicon data set from the same sample a partial 18S rRNA gene sequence that is thought to represent the new species, with high probability. A comparison of our sequence to those present in the Diat.barcode and NCBI databases showed that the sequence is closely related to, but significantly different from, that of Mayamaea terrestris. Natural saline habitats in Serbia and neighbouring countries are under anthropogenic threat and climate change, so knowing which species live here is the first step in preserving these unique habitats.

Antimicrobial Activity of Extremely Halophilic Archaea Isolated from Southern Thai Salt-Fermented Products and Solar Saltern of Pattani, Thailand

This research aimed to study the diversity and antimicrobial activity of culturable haloarchaea in soil samples of solar saltern in Pattani Province, Thailand and Southern Thai salt-fermented food. Seventy-seven extremely haloarchaea were isolated on Halophilic medium agar containing 25% NaCl at 37°C for 7-21 days. They were grouped by Amplified Ribosomal DNA Restriction Analysis (ARDRA) with two restriction enzymes, RsaI and HindIII. The ARDRA patterns illustrated 6 different Operation Taxonomic Units (OTUs). Partial 16S rRNA gene sequences (938 bp) of the representative of each OTUs were aligned with the GenBank database. The results showed that the representatives of OTU1, OTU2, OTU3, OTU4, OTU5, and OTU6 had 99-100% similarity to Halobacterium salinarum, 99-100% similarity to Halostagnicola larsenii, 99% similarity to Natronococcus sp., 99-100% similarity to Haloferax alexandrinus, 99-100% similarity to Natrialba sp. and 97% similarity to Halococcus sp., respectively. The antimicrobial activity testing of the 17 isolated haloarchaeal strains from solar saltern was performed against 5 tested strains of Hbt. salinarum and 1 strain of Natrialba sp. were isolated from fermented food. Seven (41.2%) were candidate halocin-producing strains. In addition, no phage activity was observed. The 14-day fermentation broth of Natronococcus sp. SS13 showed the highest antimicrobial activity against Hbt. salinarum PK08, BD07 and BD09 (>5,120 AU/ml).

Molecular Identification, Pathogenesis, and Life Cycle of Sarcocystis cruzi from Cattle (Bos taurus) in New Valley Governorate, Egypt.

Sarcocystis cruzi was identified by molecular methods from an intermediate host, cattle (Bos taurus), in El-Kharga, New Valley Governorate, Egypt, and its life cycle and pathogenicity were studied in the final host, dogs (Canis familiaris). 600 slaughtered cattle aged 6-8 years (480/120 males/females) were included. In addition, three laboratory-bred, coccidian-free puppies aged 2-3 months were fed infected bovine muscles to locate the definitive host and analyze sporogony. 18S rRNA-specific gene primers were used for DNA amplification from esophageal muscles. These polymerase chain reaction (PCR) amplicons were subjected to restriction fragment length polymorphism (RFLP) and molecular sequence analysis. Infection was detected in 78.8% (473/600; 95% CI, 75.56-82.11%). Histopathological examination of esophageal muscles showed oval- to spherical-shaped cysts, 96.7 μm wide by 326.9 μm long; cysts in cardiac muscles were ovoid and smaller. Infected puppies began shedding sporocysts in feces 7 days post-inoculation and showed distorted organ architecture, severe cellular damage, and inflammatory lesions in liver, kidney, esophagus, and stomach. Three oocysts with different shapes and sizes were identified. Partial 18S rRNA gene sequences of isolated New Valley sarcocysts were identical to S. cruzi isolated from different areas, verifying their genetic relatedness. Our analysis suggests that S. cruzi is the most prevalent in slaughtered cattle in New Valley Governorate, Egypt.

Comparison of Atmospheric and Lithospheric Culturable Bacterial Communities from Two Dissimilar Active Volcanic Sites, Surtsey Island and Fimmvörðuháls Mountain in Iceland.

Surface microbes are aerosolized into the atmosphere by wind and events such as dust storms and volcanic eruptions. Before they reach their deposition site, they experience stressful atmospheric conditions which preclude the successful dispersal of a large fraction of cells. In this study, our objectives were to assess and compare the atmospheric and lithospheric bacterial cultivable diversity of two geographically different Icelandic volcanic sites: the island Surtsey and the Fimmvörðuháls mountain, to predict the origin of the culturable microbes from these sites, and to select airborne candidates for further investigation. Using a combination of MALDI Biotyper analysis and partial 16S rRNA gene sequencing, a total of 1162 strains were identified, belonging to 72 species affiliated to 40 genera with potentially 26 new species. The most prevalent phyla identified were Proteobacteria and Actinobacteria. Statistical analysis showed significant differences between atmospheric and lithospheric microbial communities, with distinct communities in Surtsey's air. By combining the air mass back trajectories and the analysis of the closest representative species of our isolates, we concluded that 85% of our isolates came from the surrounding environments and only 15% from long distances. The taxonomic proportions of the isolates were reflected by the site's nature and location.

Analysis of Partial Mitochondrial COI and 16S rRNA Gene Sequences Variation of Pagurus brachiomastus and Pagurus proximus (Decapoda: Paguridae) Populations from the South of Russian Far East

Mitochondrial COI and 16S rRNA gene partial sequences were generated for Pagurus brachiomastus from two locations and P. proximus from single location of the south of Russian Far East. Genetic variation and differentiation of P. brachiomastus Russian and South Korean (GenBank data) populations were estimated and compared on the basis of sequence data on two mitochondrial genes separately. Additionally, P. simulans from South Korea was analyzed. Results show P. brachiomastus from two locations of the south of Russian Far East and South Korea and Korean P. simulans differs at intraspecific level based on molecular markers. Median-Joining analysis indicate presence of two main groups of haplotypes, representing P. proximus and P. brachiomastus + P. simulans, respectively. These data support that P. brachiomastus and P. simulans are conspecific. Mismatch distribution analysis showed population growth for P. brachiomastus at the south of Russian Far East. Sequence pairwise variations of both molecular markers appear differently in two far eastern samples of P. brachiomastus, that can be partially explained by active gene flow through hermit crab migrations between two localities after their isolation.

Isolation and phenotypic characterization of bacteriophage SA14 with lytic- and anti-biofilm activity against multidrug-resistant Enterococcus faecalis

BackgroundAntimicrobial resistance is a growing global health concern demanding more attention and action at the international-, national- and regional levels. In the present study, bacteriophage was sought as a potential alternative to traditional antibiotics.ResultsVancomycin-resistant Enterococcus faecalis was isolated from a urine sample. Partial 16S rRNA-gene sequencing and VITEK®2 system were employed for its identification, biochemical characterization, and antibiotic susceptibility testing. The isolate was resistant to eight antibiotics (out of 11): vancomycin, gentamicin (high-level synergy), streptomycin (high-level synergy), ciprofloxacin, levofloxacin, erythromycin, quinupristin/dalfopristin, and tetracycline. Bacteriophage SA14 was isolated from sewage water using the multidrug-resistant isolate as a host. Transmission electron micrographs revealed that phage SA14 is a member of the Siphoviridae family displaying the typical circular head and long non-contractile tail. The phage showed characteristic stability to a wide range of solution pH and temperatures, with optimal stability at pH 7.4 and 4 °C, while showing high specificity toward their host. Based on the one-step growth curve, the phage's latent period was 25 min, and the burst size was 20 PFU/ml. The lytic activity of phage SA14 was evaluated at various multiplicities of infection (MOI), all considerably suppressed the growth of the host organism. Moreover, phage SA14 displayed a characteristic anti-biofilm activity as observed by the reduction in adhered biomass and -viable cells in the pre-formed biofilm by 19.1-fold and 2.5-fold, respectively.ConclusionPhage therapy can be a valuable alternative to antibiotics against multi-drug resistant microorganisms.

Three chigger species (Trombidiformes: Trombiculidae) co-parasitizing a domestic cat in Brazil, including pathogen monitoring

ABSTRACT The ecology of chigger mites has many unknown aspects, even with several recent studies addressing the taxonomy and systematics of this group. In Brazil, the chigger species recorded coparasitizing vertebrates include opossums in the Pernambuco State, lizards and rodents in the Piauí State, and rodents in the São Paulo State. Chiggers collected on a domestic cat, Felis catus Linnaeus, 1758 (Mammalia: Felidae), from Caxias do Sul, Rio Grande do Sul State, were sent to the Acarological Collection of Instituto Butantan (IBSP) to be identified. The species were identified as eight larvae of Eutrombicula tinami, two larvae of Parasecia valida Brennan, 1969, and two specimens of the genus Eutrombicula Ewing, 1938, which have been described here as Eutrombicula bassinii n. sp. Additionally, we amplified partial 18S rRNA gene sequences for E. tinami and E. bassinii n. sp. However, the attempts to amplify fragments of the gltA gene of Rickettsia were unsuccessful. The present study reports the coparasitism in a domesticated feline with these three species, the second record of E. tinami, the first record P. valida to the Rio Grande do Sul State, and the description of E. bassinii n. sp. http://www.zoobank.org/urn:lsid:zoobank.org:act:D0D6EEAB-DDB6-49F0-9651-4AFB96D4D588

First report of rice bacterial leaf blight and grain discolouration caused by Pantoea agglomerans in India

A disease survey was carried out in Tamil Nadu, India during 2021 to assess the prevalence of diseases in major rice-growing areas and characterise the pathogens associated with infected plants. During the survey, rice plants at the seedling to grain-filling stage showing water-soaked, elongated, light brown lesions in the leaf margins and tips (Figure 1) were collected along with grains in a polythene bag, labelled and sealed. Leaves showing yellowing and drying from tips and margins were cut into small pieces, surface-sterilised in 1% sodium hypochlorite for one minute, rinsed three times with sterile distilled water and then allowed to air dry. After drying, the tissues were soaked for 30 minutes at room temperature in 0.5 ml of sterile distilled water and then a loop full of suspension was streaked onto nutrient agar (NA) medium. For the isolation of pathogen from infected seed, discoloured grain samples were surface sterilised in 3% sodium hypochlorite for one minute, rinsed thrice with sterile distilled water and allowed to air dry. Surface-sterilised grains were placed in a Petri plate containing NA medium. These plates were incubated at 28°C for one to two days. Upon incubation, straw to yellow-coloured colonies emerged. The colonies were further purified through single colony isolation on NA. The purified colonies were yellow in colour, translucent and undulate. (Figure 2.) The colonies exhibited umbonate surfaces with slightly concave centres (Al-Kidsawey et al., 2020; Delétoile et al., 2009). The pathogenicity of the isolates was demonstrated by inoculation of isolates on rice seedlings (cv. Co 39). For this, bacterial isolates were incubated in nutrient broth for 48 hours at 27°C. The bacterial suspension was adjusted to a concentration of 109 cfu/ml by adding sterile water. The bacterium was inoculated by cutting the tip (about 3 to 4 cm) of the fully expanded uppermost leaf with scissors and dipping in the bacterial suspension. Controls used sterile distilled water instead of the bacterial suspension. The inoculated plants were covered with polythene sheet overnight and incubated under laboratory conditions. The inoculated leaves developed progressive necrosis with colour shifts from pale green to brown within 3 to 14 days post inoculation, whereas the control plants exhibited no symptoms. Koch's postulates were fulfilled by isolating the bacterium on NA which produced yellow, flat to convex translucent colonies after 48 hours incubation. The bacterium was further subjected to biochemical tests using the KB002 HiAssorted Biochemical Test Kit (Himedia, Germany). The bacterium showed a positive reaction for citrate utilisation, phenylalanine deamination, nitrate reduction, glucose and arabinose utilization. It showed a negative reaction to lysine utilisation, ornithine utilization, urease, H2S production, adonitol, lactose and sorbitol utilization. Based on the morphological characters and biochemical tests, the bacterium was identified as a Pantoea sp. To further characterise the pathogen, DNA extraction was done, and a PCR assay was performed using universal primers 27F and 1492R targeting the 16S rRNA gene. Sequencing of the amplicon revealed that the bacterial isolates had 93.7% identity with Pantoea agglomerans type strain SGAir0210 (GenBank Accession No. CP028033.1). The partial 16S rRNA gene sequences of two strains of P. agglomerans were deposited in GenBank (OP784578 and OP784743). Pantoea agglomerans causing bacterial leaf blight and glume discolouration has been reported from Brazil, South Korea, Turkey and Venezuela (Aksoy & Boluk, 2019; Carrer Filho et al., 2018; González et al., 2015; Lee et al., 2010). Pantoea agglomerans also causes infection in humans (Al-Kidsawey et al., 2020; Delétoile et al., 2009)). Bacterial leaf blight in rice caused by P. stewartii subsp. indologenes has been reported from Tamil Nadu, India (Vinodhini et al., 2017). However, to our knowledge, this is the first report of P. agglomerans causing rice leaf blight and grain discolouration in India. Further studies are needed to ascertain the prevalence of P. agglomerans in commercial rice cultivars in other parts of India. We gratefully acknowledge the facilities provided by DST-FIST Lab, Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India.

First molecular report of Babesia gibsoni infection in pet dogs in Hunan province, subtropical China.

Canine babesiosis caused by Babesia spp. is a noteworthy tick-borne zoonotic disease of domestic dogs and wild canids. In present study, a total of 556 blood samples were randomly collected from pet dogs in eight cities of Hunan province, subtropical China. Genomic DNA was extracted and Babesia DNA was detected by amplification of partial 18S rRNA gene sequences. A total of 56 (10.1%) blood samples were tested positive for Babesia species. Sequence analysis showed that 29 dogs (5.2%) were positive for B. gibsoni, and other 27 dogs for B. vogeli (4.9%). The age and health status were considered as important risk factors for B. gibsoni and B. vogeli infections in pet dogs in this study (P<0.05). Phylogenetic analysis showed that the examined positive samples were highly clustered in the same branch with B. gibsoni and B. vogeli, respectively. This is the first molecular report of B. gibsoni infection in pet dogs in Hunan province, subtropical China. Our finding has provided a guide for the control of dog babesiosis in China and elsewhere.

Utility evaluation of two molecular methods for Leptospira spp. typing in human serum samples

Most of the available genotyping methods were applied and evaluated in Leptospira isolates and only few of them in a relevant sample size of blood specimens but not of sera. The objective of this study was to evaluate the utility of one partial 16S rRNA gene sequencing assay (16S rRNA) and an optimized. Multilocus sequence typing scheme (MLST) for Leptospira typing directly in serum samples. Confirmed leptospirosis patients (n = 228) from Argentina (2005–2016) were randomly included. Septicemic-phase serum samples (n = 228) were studied by two genotyping methods. Available immune-phase serum samples of the included patients (n = 159) were studied by MAT to compare serological and molecular results. In culture-proven cases (n = 8), genotyping results between clinical samples and isolates were compared. Typing success rate (TSR) was 21.9% for 16S rRNA and 11.4% for MLST (full allelic profile) and a positive trend in both TSR during the study period was observed. Two species (L. interrogans and L. borgpertesenii) were identified by both methods and MLST assigned 8 different STs. The probable serogroups identified by MLST were coincident with the presumptive infecting serogroups identified by MAT, but with different frequencies. The three serogroups (Canicola, Sejroe and Icterohaemorrhagiae) most frequently identified by MAT were also genotyped by MLST. Typing results via 16S rRNA and MLST in clinical samples and isolates of culture-proven cases, were consistent except for one case. Performance of partial 16S rRNA gene sequencing assay and the optimized MLST scheme directly in sera may increase and improve the knowledge about species and serogroups causing human leptospirosis, especially in countries with low rates of culture sample collection or Leptospira isolation.