Partial 16S rRNA Gene Sequences Research Articles

Isolation and characterization of a heavy metal- and antibiotic-tolerant novel bacterial strain from a contaminated culture plate of Chlamydomonas reinhardtii, a green micro-alga.

Background:Chlamydomonas reinhardtii, a green micro-alga, is normally cultured in laboratories in Tris-Acetate Phosphate (TAP), a medium which contains acetate as the sole carbon source. Acetate in TAP can lead to occasional bacterial and fungal contamination. We isolated a yellow-pigmented bacterium from a Chlamydomonas TAP plate. It was named Clip185 based on the Chlamydomonas strain plate it was isolated from. In this article we present our work on the isolation, taxonomic identification and physiological and biochemical characterizations of Clip185. Methods: We measured sensitivities of Clip185 to five antibiotics and performed standard microbiological tests to characterize it. We partially sequenced the 16S rRNA gene of Clip185. We identified the yellow pigment of Clip185 by spectrophotometric analyses. We tested tolerance of Clip185 to six heavy metals by monitoring its growth on Lysogeny Broth (LB) media plates containing 0.5 mM -10 mM concentrations of six different heavy metals. Results: Clip185 is an aerobic, gram-positive rod, oxidase-negative, mesophilic, alpha-hemolytic bacterium. It can ferment glucose, sucrose and mannitol. It is starch hydrolysis-positive. It is very sensitive to vancomycin but resistant to penicillin and other bacterial cell membrane- and protein synthesis-disrupting antibiotics. Clip185 produces a C50 carotenoid, decaprenoxanthin, which is a powerful anti-oxidant with a commercial demand. Decaprenoxanthin production is induced in Clip185 under light. NCBI-BLAST analyses of the partial 16S rRNA gene sequence of Clip185 revealed a 99% sequence identity to that of Microbacterium binotii strain PK1-12M and Microbacterium sp. strain MDP6. Clip185 is able to tolerate toxic concentrations of six heavy metals. Conclusions: Our results show that Clip185 belongs to the genus Microbacterium. In the future, whole genome sequencing of Clip185 will clarify if Clip185 is a new Microbacterium species or a novel strain of Microbacterium binotii, and will reveal its genes involved in antibiotic-resistance, heavy-metal tolerance and regulation of decaprenoxanthin biosynthesis.

Gulosibacter hominis sp. nov.: a novel human microbiome bacterium that may cause opportunistic infections

We present genomic, phylogenomic, and phenotypic taxonomic data to demonstrate that three human ear isolates represent a novel species within the genus Gulosibacter. These isolates could not be identified reliably using MALDI-TOF mass spectrometry during routine diagnostic work, but partial 16S rRNA gene sequence analysis revealed that they belonged to the genus Gulosibacter. Overall genomic relatedness indices between the draft genome sequences of the three isolates and of the type strains of established Gulosibacter species confirmed that the three isolates represented a single novel Gulosibacter species. A biochemical characterisation yielded differential tests between the novel and established Gulosibacter species, which could also be differentiated using MALDI-TOF mass spectrometry. We propose to formally classify these three isolates into Gulosibacter hominis sp. nov., with 401352-2018T (= LMG 31778T, CCUG 74795T) as the type strain. The whole-genome sequence of strain 401352-2018T has a size of 2,340,181bp and a G+C content of 62.04mol%. A Gulosibacter pangenome analysis revealed 467 gene clusters that were exclusively present in G. hominis genomes. While these G. hominis specific gene clusters were enriched in several COG functional categories, this analysis did not reveal functions that suggested a role in the human microbiome, nor did it explain the occurrence of G. hominis in ear infections. The absence of acquired antimicrobial resistance determinants and virulence factors in the G. hominis genomes, and an analysis of publicly available 16S rRNA gene sequences and 16S rRNA amplicon sequencing data sets suggested that G. hominis is a member of the human skin microbiota that may occasionally be involved in opportunistic infections.

First Molecular Characterisation of Blastocystis from Experimental Rats in Turkey and Comparison of the Frequencies Between Obese and Non-obese Groups

Blastocystis is a zoonotic protozoan that infects a wide range of animals, including humans and rodents. This study aimed to determine the frequency and subtype distribution of Blastocystis in laboratory rats at a laboratory animal facility in Turkey. This study included 54 male Sprague-Dawley rats from Aydın Adnan Menderes University Laboratory Animal Center. Among these rats, 30 were fed with high-fat diet (obese group) and the remaining 24 received standard chow (non-obese group). Blastocystis positivity was determined with amplification of small subunit 18S rRNA gene following their nucleic acid extraction from faecal samples. Subtypes were detected by submitting the partial 18S rRNA gene sequences to the database (pubmlst. Blastocystis infection was detected in 33 (61.1%) of 54 laboratory rats. The frequency of Blastocystis was significantly different between obese and non-obese rats (p<0.05), with 43.3% and 83.3%, respectively. When referred to the database, exact matches were identified with Blastocystis subtype 4 (ST4) for all isolates. In the phylogenetic analysis of the partial 18S rDNA sequence, the sequence was closely clustered with reference ST4 subtypes from other countries, including China, Japan, United Kingdom and Czech Republic. This study revealed the high rate of Blastocystis colonisation in laboratory rats, posing a risk for human transmission. The comparison of obese and non-obese groups supported the idea that Blastocystis might be an indicator of healthy gut flora. The detection of ST4 in all rats agreed with previous reports of the predominance of this subtype in rodents.

New record of bioluminescence inOdontosylliscf.australiensis(Annelida: Syllidae: Eusyllinae) in Japan

AbstractBioluminescence is widespread in the marine environment. The bioluminescence of some species of the firewormOdontosyllis(Annelida: Syllidae: Eusyllinae) has been well studied, although the presence or absence of bioluminescence in most species of this genus is yet to be revealed. The bioluminescent worms were observed after sunset around the new moon day in July and October 2020 and in July to October 2021 in Nagasaki Prefecture, Japan. Molecular phylogenetic analysis based on two mitochondrial and one nuclear gene sequence showed that the worms were closely related toOdontosyllis australiensis, but the partial 16S rRNA gene sequences differed by 2% between those of the Japanese and Australian material. Because only epitokes, i.e. morphologically modified sexually mature worms, were collected, further studies on morphological characters of atokes would be required in the future. We therefore tentatively refer to them asOdontosylliscf.australiensis. Molecular phylogenetic analysis also showed that known bioluminescentOdontosyllisspecies belong to various lineages.

Molecular detection of piroplasmids in synanthropic rodents, marsupials, and associated ticks from Brazil, with phylogenetic inference of a putative novel Babesia sp. from white-eared opossum (Didelphis albiventris).

The order Piroplasmida encompasses tick-borne pathogens of veterinary and medical importance positioned in two main families: Babesiidae and Theileriidae. Even though previous studies carried out in Brazil recorded the occurrence of piroplasmid species circulating in small mammals, 18S RNA gene sequences were only partially sequenced, preventing the assessment of their phylogenetic positioning. The current study aimed to detect and characterize, using morphological, molecular, and bioinformatic approaches, piroplasmids from wild mammals and associated ticks sampled in Central-Western Brazil. Out of 67 Didelphis albiventris sampled, 22 (16.4%) were positive for piroplasmids by PCR. In contrast, none of the 48 small rodents and 14 capybaras (Hydrochoerus hydrochaeris) was PCR-positive. Four Amblyomma dubitatum ticks-one from Rattus rattus, one from H. hydrochaeris, and two from D. albiventris-out of 114 Amblyomma spp. DNA samples were positive for piroplasmids by PCR. The phylogenetic inference performed using the near-complete 18S rRNA gene positioned the putative novel piroplasmid species detected in D. albiventris and associated A. dubitatum ticks near to Babesia sensu lato clade (Western group-cluster III) and distant from the Australian marsupial-associated piroplasms. Phylogenetic inferences based on two additional molecular markers, namely hsp-70 and cox-1, supported the near-complete 18S rRNA gene phylogenetic inference. Finally, the partial 18S rRNA gene sequences detected in ticks from rodents (R. rattus and H. hydrochaeris) showed 97.2-99.4% identity with the Piroplasmida previously detected in a capybara from Brazil, raising evidence that a still uncharacterized piroplasmid species has been identified in the capybara, the largest rodent species from South America.

Isolation and characterization of a heavy metal- and antibiotic-tolerant novel bacterial strain from a contaminated culture plate of Chlamydomonas reinhardtii, a green micro-alga.

Background:Chlamydomonas reinhardtii, a green micro-alga, is normally cultured in laboratories in Tris-Acetate Phosphate (TAP), a medium which contains acetate as the sole carbon source. Acetate in TAP can lead to occasional bacterial and fungal contamination. We isolated a yellow-pigmented bacterium from a Chlamydomonas TAP plate. It was named Clip185 based on the Chlamydomonas strain plate it was isolated from. In this article we present our work on the isolation, taxonomic identification and physiological and biochemical characterizations of Clip185.Methods: We measured sensitivities of Clip185 to five antibiotics and performed standard microbiological tests to characterize it. We partially sequenced the 16S rRNA gene of Clip185. We identified the yellow pigment of Clip185 by spectrophotometric analyses. We tested tolerance of Clip185 to six heavy metals by monitoring its growth on Lysogeny Broth (LB) media plates containing 0.5 mM -10 mM concentrations of six different heavy metals.Results: Clip185 is an aerobic, gram-positive rod, oxidase-negative, mesophilic, alpha-hemolytic bacterium. It can ferment glucose, sucrose and mannitol. It is starch hydrolysis-positive. It is very sensitive to vancomycin but resistant to penicillin and other bacterial cell membrane- and protein synthesis-disrupting antibiotics. Clip185 produces a C50 carotenoid, decaprenoxanthin, which is a powerful anti-oxidant with a commercial demand. Decaprenoxanthin production is induced in Clip185 under light. NCBI-BLAST analyses of the partial 16S rRNA gene sequence of Clip185 revealed a 99% sequence identity to that of Microbacterium binotii strain PK1-12M and Microbacterium sp. strain MDP6. Clip185 is able to tolerate toxic concentrations of six heavy metals.Conclusions: Our results show that Clip185 belongs to the genus Microbacterium. In the future, whole genome sequencing of Clip185 will clarify if Clip185 is a new Microbacterium species or a novel strain of Microbacterium binotii, and will reveal its genes involved in antibiotic-resistance, heavy-metal tolerance and regulation of decaprenoxanthin biosynthesis.

Presence of Lactic Acid Bacteria in the Intestinal Tract of the Mediterranean Trout (Salmo macrostigma) in Its Natural Environment.

Knowledge of the composition of the gut microbiota in freshwater fish living in their natural habitat has taxonomic and ecological importance. Few reports have been produced on the composition of the gut microbiota and on the presence of LAB in the intestines of freshwater fish that inhabit river environments. In this study, we investigated the LAB community that was present in the gastrointestinal tract (GIT) of Mediterranean trout (Salmo macrostigma) that colonized the Biferno and Volturno rivers of the Molise region (Italy). The partial 16S rRNA gene sequences of these strains were determined for the species-level taxonomic placement. The phylogenetic analysis revealed that the isolated LABs belonged to seven genera (Carnobacterium, Enterococcus, Lactobacillus, Lactiplantibacillus, Vagococcus, Lactococcus, and Weissella). The study of the enzymatic activities showed that these LABs could contribute to the breakdown of polysaccharides, proteins, and lipids. In future studies, a greater understanding of how the LABs act against pathogens and trigger the fish immune response may provide practical means to engineer the indigenous fish microbiome and enhance disease control and fish health.

Isolation and characterization of a heavy metal- and antibiotic-tolerant novel bacterial strain from a contaminated culture plate of Chlamydomonas reinhardtii, a green micro-alga.

Background:Chlamydomonas reinhardtii, a green micro-alga, is normally cultured in laboratories in Tris-Acetate Phosphate (TAP), a medium which contains acetate as the sole carbon source. Acetate in TAP can lead to occasional bacterial and fungal contamination. We isolated a yellow-pigmented bacterium from a Chlamydomonas TAP plate. It was named Clip185 based on the Chlamydomonas strain plate it was isolated from. In this article we present our work on the isolation, taxonomic identification and physiological and biochemical characterizations of Clip185. Methods: We measured sensitivities of Clip185 to five antibiotics and performed standard microbiological tests to characterize it. We partially sequenced the 16S rRNA gene of Clip185. We identified the yellow pigment of Clip185 by spectrophotometric analyses. We tested tolerance of Clip185 to six heavy metals by monitoring its growth on Lysogeny Broth (LB) media plates containing 0.5 mM -10 mM concentrations of six different heavy metals. Results: Clip185 is an aerobic, gram-positive rod, oxidase-negative, mesophilic, alpha-hemolytic bacterium. It can ferment glucose, sucrose and mannitol. It is starch hydrolysis-positive. It is very sensitive to vancomycin but resistant to penicillin and other bacterial cell membrane- and protein synthesis-disrupting antibiotics. Clip185 produces a C50 carotenoid, decaprenoxanthin, which is a powerful anti-oxidant with a commercial demand. Decaprenoxanthin production is induced in Clip185 under light. NCBI-BLAST analyses of the partial 16S rRNA gene sequence of Clip185 revealed a 99% sequence identity to that of Microbacterium binotii strain PK1-12M and Microbacterium sp. strain MDP6. Clip185 is able to tolerate toxic concentrations of six heavy metals. Conclusions: Our results show that Clip185 belongs to the genus Microbacterium. In the future, whole genome sequencing of Clip185 will clarify if Clip185 is a new Microbacterium species or a novel strain of Microbacterium binotii, and will reveal its genes involved in antibiotic-resistance, heavy-metal tolerance and regulation of decaprenoxanthin biosynthesis.

Molecular Identification and Genotyping of Theileria Orientalis Type 3 (Buffeli) Isolated from Cattle Using Nested-PCR Assay in Guilan Province, Iran.

Protozoan parasites of the genus Theileria are tick-borne parasites that have been found in many species of mammals. More than a dozen species of Theileria have been found in cattle, water buffalo, sheep, and goats. Theileria orientalis is a non-pathogenic blood protozoan parasite that was detected and identified during a regular investigation of piroplasmida infection in indigenous cattle in the spring of 2019 in Northern Provinces of Iran. In total, 92 blood samples were collected from different areas of Guilan and Mazandaran Provinces, Iran during the spring. The Giemsa stained blood smears did not show any parasitic infection; however, T. orientalis was identified by 18S rRNA gene polymerase chain reaction (PCR) and DNA sequencing. The specific sequenced DNA for T. orientalis was registered in GenBank under the accession number MN453385. The partial 18S rRNA gene sequence of the obtained DNA showed 100% nucleotide identity with reference sequences for the T. orientalis that have been registered from Europe, Africa, and Asia. Additionally, molecular phylogenetic studies have shown that T. orientalis Iran GC98-01 isolate belongs to nonpathogenic T. orientalis type 3 (buffeli). In this study, the indigenous Bos indicus cattle were detected as asymptomatic carriers of Theileria spp. infection. Here, we identified and genotyped T. orientalis for the first time as T. orientalis type 3 (buffeli) in Iran using molecular phylogenetic analysis and registered the 18S rRNA gene sequence of the T. orientalis GC98-01 isolate in GenBank. Moreover, rare T. annulata infection was detected in cattle using semi-nested PCR in Mazandaran (Miankaleh peninsula). The T. orientalis can be differentiated from other Theileria and Babesia haemoprotozoan parasites by specific molecular assays.

Isolation and molecular identification of thermophilic bacteria from litter of mount Galunggung hot spring, Tasikmalaya, Indonesia

Thermophilic bacteria from litter deposited at Cipanas hot spring of mount Galunggung, Tasikmalaya, West Java, Indonesia, were isolated using a culture-dependent approach. Medium International Streptomyces Project (ISP) 1 solidified with gellan gum was used as an isolation medium. Isolation plates were incubated at 50°C for three weeks. The physicochemical analysis showed that the hot spring has a neutral pH and temperatures ranging from 50−56°C. A total of 16 bacterial isolates were obtained and purified. The analysis of partial 16S rRNA gene sequences data and phylogenetic analyses showed that they belong to Firmicutes and Proteobacteria phyla. The majority of bacterial isolates are spore-forming bacteria. The molecular identification based on the 16S rRNA gene sequence data showed that they belong to Aneurinibacillus thermoaerophilus (1); Brevibacillus gelatini (7); Bacillus licheniformis (1); Chelatococcus composti (1); Laceyella sacchari (2); Paenibacillus barengoltzii (1); and Pseudoxanthomonas taiwanensis (3). Percentage sequence similarities to their closest taxa were 91 to 99%. Phylogenetic analysis shed a light to the detection of candidates of novel taxa from litter of mount Galunggung hot spring.

Prevalence, Antibiogram and Genetic Characterization of Listeria monocytogenes from Food Products in Egypt.

World Health Organization classified Listeria monocytogenes as a major notable foodborne pathogen associated with high mortality and hospitalization. The study reports the prevalence, antibiogram, virulence determination and genetic characterization of L. monocytogenes from different food products. A total of 250 food samples, fifty samples each from raw milk, ice cream, minced meat, fish fillet and sausage were collected from the Menoufiya governorate in Egypt. L. monocytogenes was detected in 17 (6.8%) of the tested food samples including minced meat (14%), fish fillet (8%), sausage (6%) and raw milk (6%). The antimicrobial susceptibility assay of 17 L. monocytogenes isolates against seventeen antibiotics belonging to eight antibiotics classes revealed a high susceptibility to norfloxacin (82.3%), amoxicillin-clavulanic acid (76.4%), cefotaxime (70.5%), erythromycin (64.6%), amoxicillin (64.6%), gentamicin (58.7%) and vancomycin (58.7%). While, high resistance was observed against oxytetracycline (76.4%), trimethoprim-sulfamethoxazole (76.4%), chloramphenicol (70.5%), doxycycline (64.6%), levofloxacin (41.2%) and azithromycin (41.2%). Of note, all L. monocytogenes isolates were multidrug-resistant. The multiplex PCR successfully amplified L. monocytogenes in all tested isolates. Screening of the five virulence-related genes revealed the hlyA and iap as the most prevalent genes followed by actA gene, however, the inlA and prfA genes were not detected in any of the studied isolates. The partial 16S rRNA gene sequencing of three L. monocytogenes isolates showed a high nucleotide similarity (99.1–99.8%) between the study isolates and various global clones, and phylogenetic analysis clustered these L. monocytogenes strains with other Listeria species including L. welshimeri, L. seeligeri and L. innocua. This study demonstrates the impact of L. monocytogenes as a major contaminant of various food products and suggests more attention to the awareness and hygienic measures in the food industry.

A comparative GC-MS analysis of bioactive secondary metabolites produced by halotolerant Bacillus spp. isolated from the Great Sebkha of Oran.

The reemergence of infectious diseases and resistant pathogens represents a serious problem for human life. Hence, the screening for new or alternative antimicrobial compounds is still urgent. Unusual ecosystems such as saline habitats are considered promising environments for the purposes of isolating bacterial strains able to produce potent natural products. The aim of this study is the identification of bioactive compounds biosynthesized by three halotolerant strains isolated from the Sebkha of Oran (Algeria) using gas chromatography coupled to mass spectrometry. Primary screening investigation of antimicrobial activities were performed against reference bacterial and fungal strains and revealed a broad-spectrum activity. Secondary metabolite extraction was carried out using ethyl acetate and chloroform. Crude extracts were tested for bioactivity using the disc diffusion method and subjected to GC-MS analysis. The extracts showed an important inhibitory effect against all tested strains. Fifty-six compounds were identified; they include tert-butyl phenol compounds, fatty acid methyl esters due to the methylation procedure, hydrocarbons, aldehydes, benzoquinones, pyrrols, and terpenes. Literature reports such compounds to have wide biological and pharmaceutical applications. The molecular identification of the three isolates was achieved using the 16S-23S rRNA gene intergenic spacer region (ITS) and 16S rRNA sequencing. Partial 16S rRNA gene sequencing showed very high similarity with many species of Bacillus. This study provided insights on the potential of halotolerant Bacillus as drug research target for bioactive metabolites. The findings suggest that the Great Sebkha of Oran is a valuable source of strains exhibiting variety of beneficial attributes that can be utilized in the development of biological antibiotics.

Gibbsiella quercinecans as new pathogen involved in bacterial canker of Russian olive.

Russian olive (Elaeagnus angustifolia) is an economically important ornamental and crop plant. It has a wide range of biologically active compounds and is regarded as a unique medicinal plant with multiple applications. Members of the order Enterobacteriales isolated from trees are often associated with bacterial canker. During the growing season from 2017 to 2020, forty Gram-negative bacterial strains were isolated from Russian olive trees infected with cankers showing symptoms such as distinctive lesions on the trunk and branches, decline, weakness of trees, cracked bark, depressed brown to black lesions accompanied by exudation of gum, in the Kerman and Mazandaran Provinces of Iran. We used a polyphasic approach to identify and characterize these strains. Using a multilocus sequence analysis approach of four housekeeping loci, namely atpD, rpoB, gyrB, infB, and partial 16S rRNA gene sequences, isolates were identified as Gibbsiella quercinecans. These results were further supported by phenotypic and biochemical tests. Results of the biochemical, physiological and phenotypic experiments, indicated that the isolates are members of the Enterobacteriales and within the genus Gibbsiella. Pathogenicity of the G. quercinecans isolates was confirmed by fulfillment of Koch's postulates. To our knowledge, this is the first report of G. quercinecans as the causal agent of bacterial canker of Russian olive trees.

Screening and Identification of Lipopeptide Biosurfactants Produced by Two Aerobic Endospore-Forming Bacteria Isolated from Mfabeni Peatland, South Africa.

Two aerobic endospore-forming bacteria (AEFB), isolates SAB19 and SAD18, capable of biosurfactant production were isolated from a sediment core sampled from Mfabeni peatland, St Lucia, KwaZulu-Natal, South Africa. The isolates were screened for biosurfactant activity using drop collapse assay, hemolysis assay, oil spreading assay, emulsification, and surface tension measurement. The effect of environmental parameters--temperature [35 - 100 °C], pH [3.0 - 10.0], and salinity [0.5 - 15%]--on biosurfactant stability was also determined. Ultra-performance liquid chromatography in conjunction with electrospray ionization time-of-flight mass spectrometry (UPLC ESI-TOF MS) analysis revealed that both isolates produced surfactin isomers and a common mass peak of m/z 1326.1 that was ascribed to a precursor of the antibiotic plantazolicin (PZN). Isolate SAD18 was also found to produce the lipopeptides fengycin and iturin. Taxonomic classification based on partial 16S rRNA gene sequencing revealed that isolates SAB19 and SAD18 belonged to the Brevibacillus and Bacillus genera, respectively. The GenBank accession numbers obtained for SAB19 and SAD18 are MW429226 and MW441217. Biosurfactant extracts from isolate SAD18 exhibited the greatest level of surfactant activity and stability over the range of environmental parameters tested. Although no novel biosurfactants were identified, it was confirmed that the peatland environment represents an untapped source of microbial diversity with potential biotechnological applications.

Seasonal Distribution of Cyanobacteria in Three Urban Eutrophic Lakes Results from an Epidemic-like Response to Environmental Conditions.

Cyanobacterial communities of three co-located eutrophic sandpit lakes were surveyed during 2016 and 2017 over season and depth using high-throughput DNA sequencing of the 16S rRNA gene. All three lakes were stratified except during April 2017 when the lakes were recovering from a strong mixing event. 16S rRNA gene V4 sequences were parsed into operational taxonomic units (OTUs) at 99% sequence identity. After rarefaction of 139 samples to 25,000 sequences per sample, a combined total of 921,529 partial 16S rRNA gene sequences were identified as cyanobacteria. They were binned into 19,588 unique cyanobacterial OTUs. Of these OTUs, 11,303 were Cyanobium. Filamentous Planktothrix contributed 1537 and colonial Microcystis contributed 265. The remaining 6482 OTUs were considered unclassified. For Planktothrix and Microcystis one OTU accounted for greater than 95% of the total sequences for each genus. However, in both cases the non-dominant OTUs clustered with the dominant OTUs by date, lake, and depth. All Planktothrix OTUs and a single Cyanobium OTU were detected below the oxycline. All other Cyanobium and Microcystis OTUs were detected above the oxycline. The distribution of Cyanobium OTUs between lakes and seasons can be explained by an epidemic-like response where individual OTUs clonally rise from a diverse hypolimnion population when conditions are appropriate. The importance of using 99% identity over the more commonly used 97% is discussed with respect to cyanobacterial community structure. The approach described here can provide another valuable tool for assessing cyanobacterial populations and provide greater insight into the controls of cyanobacterial blooms.

Molecular characterization of the bacterial communities present in sheep's milk and cheese produced in South Brazilian Region via 16S rRNA gene metabarcoding sequencing

Sheep's milk and cheese represent unexplored reservoirs of microorganisms' genetic and metabolic diversity. This study aimed to characterize the microbial communities of milk and cheese from sheep of Lacaune breed, produced in the South Region of Brazil. Milk samples (n = 15) from three dairy farms and four types of cheese (fresh, colonial, feta-type, and pecorino-type; n = 20) were subjected to partial 16S rRNA gene sequencing. Streptococcus spp. and Lactobacillus spp. were the dominant taxa (core microbiota) of cheeses, while Phyllobacterium spp. and Staphylococcus spp. were the most prevalent genera in milk. Regarding milk samples, no differences in alpha diversity were observed between the analyzed farms. However, beta diversity analysis revealed that milk collected on Farm 1 differed from the others. These differences may be associated with sheep feeding, mammary gland diseases, and the milking practices used. Upon analyzing cheese samples, significant differences in both alpha and beta diversities were observed between the different cheese types, suggesting that processing and maturation conditions are important for shaping cheese microbiota. Notably, other bacterial groups—including decomposers and potentially pathogenic microorganisms to humans—were observed in some of the analyzed cheeses. This study expands our knowledge of the bacterial composition of sheep's milk and cheese found in different geographic regions.

First report of a Jujube witches’‐broom phytoplasma (16SrV) strain associated with witches’‐broom and little leaf disease of Solanum melongena in India

Solanum melongena (aubergine or brinjal; family Solanaceae) is a popular vegetable grown extensively in India, Bangladesh, Pakistan, China, Japan, Philippines, Egypt, France, Italy, and the United States (Chadha et al., 1993). Brinjal's cultivation is hampered by the Brinjal little leaf (BLL) phytoplasma. A phytoplasma survey was conducted in various brinjal production areas of Hoshangabad in Central India (Figure 1) during May 2019. Phytoplasma-like symptoms such as proliferation of axillary shoots, witches’- broom, excessive branching, little leaf and the absence of flowers and fruits when compared to symptomless brinjal plants were observed (Figure 2). For molecular detection of phytoplasma, total DNA was isolated from diseased and symptomless leaf samples (100 mg). The phytoplasma 16S rRNA gene was amplified by direct PCR using P1/P6 primers (Deng & Hiruki, 1991) and nested PCR with universal primers R16F2n/R16R2 (Gundersen & Lee, 1996), producing amplicons of the expected size (c. 1.2 kb) for the diseased (4/4) samples but not from the symptomless plant tested. Two nested PCR amplicons were purified (Wizard SV gel extraction kit; Promega, USA) and sequenced bidirectionally (Bioinovations Pvt. Ltd., India). The consensus partial 16S rRNA gene sequences were identical and the sequence data was submitted to GenBank, Accession Nos. MW025971 (BR01) and MW025257 (BR02). BLASTn analysis showed that the sequence shared 99% sequence identity with the 16S rRNA gene sequence of Jujube witches'-broom phytoplasma (MH972556, MH972553, MH972548) and ‘Candidatus Phytoplama balanitae’ (HG937644, LT558785, MH819290), Periwinkle yellows phytoplasma (EU375835) and the Elm yellows group (16SrV). Phylogenetic analysis (MEGA v 7.1) showed the closest relationships with strains of Jujube witches'-broom phytoplasma (MH972556, H744152 and MH972548) and ‘Ca. P. balanitae’ (LT558785), a member the group 16SrV, ‘Ca. P. ulmi’ (Figure 3). Six phytoplasma ribosomal groups (16SrI, 16SrII, 16SrIII, 16SrVI, 16SrIX, 16SrXII) have been associated with S. melongena globally. BLL phytoplasmas were first reported in India in 1995 (Schneider et al., 1995). In India and Japan, a BLL phytoplasma was identified as a member of the group 16SrI, ‘Ca. P. asteris’ (Kumar et al., 2012). The group 16SrVI, ‘Ca. Phytoplasma trifolii’ was also associated with BLL symptoms in India (Yadav et al., 2015). However, this is the first report of a ‘Ca. P. ulmi’ strain (group 16SrV), associated with a witches'-broom disease of brinjal in India. This finding has a significant impact for future epidemiological studies since brinjal could be an alternative host for 16SrV phytoplasma strain which affect several other important vegetable crops cultivated in Hoshangabad, Central India. The authors would like to thank to the Head of the Department of Microbiology and Vice Chancellor, Barkatullah University, Bhopal for support and encouragement.

Benchmarking of 16S rRNA gene databases using known strain sequences.

16S rRNA gene analysis is the most convenient and robust method for microbiome studies. Inaccurate taxonomic assignment of bacterial strains could have deleterious effects as all downstream analyses rely heavily on the accurate assessment of microbial taxonomy. The use of mock communities to check the reliability of the results has been suggested. However, often the mock communities used in most of the studies represent only a small fraction of taxa and are used mostly as validation of sequencing run to estimate sequencing artifacts. Moreover, a large number of databases and tools available for classification and taxonomic assignment of the 16S rRNA gene make it challenging to select the best-suited method for a particular dataset. In the present study, we used authentic and validly published 16S rRNA gene type strain sequences (full length, V3-V4 region) and analyzed them using a widely used QIIME pipeline along with different parameters of OTU clustering and QIIME compatible databases. Data Analysis Measures (DAM) revealed a high discrepancy in ratifying the taxonomy at different taxonomic hierarchies. Beta diversity analysis showed clear segregation of different DAMs. Limited differences were observed in reference data set analysis using partial (V3-V4) and full-length 16S rRNA gene sequences, which signify the reliability of partial 16S rRNA gene sequences in microbiome studies. Our analysis also highlights common discrepancies observed at various taxonomic levels using various methods and databases.

Benchmarking of 16S rRNA gene databases using known strain sequences

16S rRNA gene analysis is the most convenient and robust method for microbiome studies. Inaccurate taxonomic assignment of bacterial strains could have deleterious effects as all downstream analyses rely heavily on the accurate assessment of microbial taxonomy. The use of mock communities to check the reliability of the results has been suggested. However, often the mock communities used in most of the studies represent only a small fraction of taxa and are used mostly as validation of sequencing run to estimate sequencing artifacts. Moreover, a large number of databases and tools available for classification and taxonomic assignment of the 16S rRNA gene make it challenging to select the best-suited method for a particular dataset. In the present study, we used authentic and validly published 16S rRNA gene type strain sequences (full length, V3-V4 region) and analyzed them using a widely used QIIME pipeline along with different parameters of OTU clustering and QIIME compatible databases. Data Analysis Measures (DAM) revealed a high discrepancy in ratifying the taxonomy at different taxonomic hierarchies. Beta diversity analysis showed clear segregation of different DAMs. Limited differences were observed in reference data set analysis using partial (V3-V4) and full-length 16S rRNA gene sequences, which signify the reliability of partial 16S rRNA gene sequences in microbiome studies. Our analysis also highlights common discrepancies observed at varioustaxonomic levels using various methods and databases.

Genotypic characterization of soil bacteria in the Umm Al-Namil Island, Kuwait.

Microflora is an integral part of soil ecosystem, in which bacteria are the largest group of soil microbes. This is a pioneer study for establishing baseline data on the diversity of soil bacteria among different regions in Kuwait. The aim is to understand biodiversity in different settings, how bacteria adapt to different niches in the environment as well as in different hosts. The identification of bacterial 16S rRNA molecules from environmental soil samples was investigated. Genomic Deoxyribonucleic acid DNA was extracted from 25 soil samples derived from five different test regions in the Umm Al-Namil Island, Kuwait. After amplification of bacterial 16S rRNA molecules by the Polymerase chain reaction PCR, the products were characterized and complex band patterns were obtained, indicating high bacterial diversity. A sample of the 16 s rRNA amplicons were sequenced in order to identify the species. The spatial distribution of bacterial taxa in the different soil samples was homogeneous, suggesting a stable and widespread community. Forty-nine isolates from Umm Al-Namil island were identified by comparative analysis of partial 16S rRNA gene sequences. Phylogenetic analysis was carried out in order to study the connection between the isolates to identify species. A large proportion of these isolates represent correspond to known or novel species within the Pseudomonus and Bacillus genera, which are common soil bacteria. Our results provided a reference for future studies to facilitate bacterial identification and ecological research in Kuwait.