16S rDNA Gene Sequence Research Articles

Identification and biological control of bacterial leaf spot disease of cucurbits

Objective: The present study was conducted for isolation and detection of the phyto-pathogen responsible for bacterial leaf spot disease of cucurbits as well as evaluation of its biological control techniques. Methods: The pathogen of the disease was isolated from infected leaf of bitter gourd and cultured on Luria-Bertani (LB) growth medium. The morphological tests and biochemical tests revealed the isolated bacteria as gram negative. Furthermore, advanced molecular technique was applied to facilitate proper detection of the isolated bacteria. The PCR of the bacterial genomic DNA using specific primers generated clear band of approximately 1450 bp in gel electrophoresis. Results: In gram staining test, the bacterial strain was found to be rod shaped and pinkish in color and Gram-negative. All the biochemical test showed positive without mannitol salt agar. The nucleotide sequencing of 16S rDNA gene of the bacterial isolate showed 86.02% similarities with the original sequence of Xanthomonas cucurbitae. In antibiotic sensitivity assay, the antibiotic cefixime showed highest 28.0±0.7 mm diameter of inhibition zone against the tested bacteria. The highest antibacterial activity was 8.0±0.5 mm zone of inhibition by of Allium sativum. Conclusion: The present findings may be benevolent for the detection, characterization and development of biological control techniques to prevent the leaf spot disease of cucurbits crops in Bangladesh. It may help to find out the specific signaling pathway techniques for plant microbes relationship for disease formation in future.

Next-Generation Sequencing of the Human Aqueous Humour Microbiome.

The microbiome of the ocular surface has been characterised, but only limited information is available on a possible silent intraocular microbial colonisation in normal eyes. Therefore, we performed next-generation sequencing (NGS) of 16S rDNA genes in the aqueous humour. The aqueous humour was sampled from three patients during cataract surgery. Air swabs, conjunctival swabs from patients as well as from healthy donors served as controls. Following DNA extraction, the V3 and V4 hypervariable regions of the 16S rDNA gene were amplified and sequenced followed by denoising. The resulting Amplicon Sequence Variants were matched to a subset of the Ribosomal Database Project 16S database. The deduced bacterial community was then statistically analysed. The DNA content in all samples was low (0-1.49 ng/µL) but sufficient for analysis. The main phyla in the samples were Acinetobacteria (48%), Proteobacteria (26%), Firmicutes (14%), Acidobacteria (8%), and Bacteroidetes (2%). Patients' conjunctival control samples and anterior chamber fluid showed similar patterns of bacterial species containing many waterborne species. Non-disinfected samples showed a different bacterial spectrum than the air swab samples. The data confirm the existence of an ocular surface microbiome. Meanwhile, a distinct intraocular microbiome was not discernible from the background, suggesting the absence of an intraocular microbiome in normal eyes.

The tongue microbiome of young patients with chronic kidney disease and their healthy mothers.

Oral microbiome plays a crucial role in the incidence and development of oral diseases. An altered intestinal microbiome has been reported in adults with chronic kidney disease (CKD). This study aimed to characterize the tongue microbiome of young patients with CKD compared to their healthy mothers to identify the influence of CKD-associated factors on resilient tongue ecosystem. Thirty patients with CKD (mean age, 14.2 years; 16 males and 14 females) and generalized gingivitis were included in the study. Swabs of the posterior tongue were collected from the patients and 21 mothers (mean age 40.8 years). Next-generation sequencing of 16S rDNA genes was employed to quantitatively characterize microbial communities. The bacterial communities were similar in terms of richness and diversity between patients and mothers (p > 0.05). In patients with CKD, 5 core phyla, 20 core genera, and 12 core species were identified. The tongue microbiome of the study participants showed no relevant CKD-associated differences compared to their mothers and appears to be a highly preserved niche in the oral cavity. Differences observed in the abundance of individual species in this study could be attributed to the age rather than CKD, even after a mean disease duration of 11 years. CKD and its associated metabolic changes appear to have no detectable impact on the resilient tongue microbiome observed in young patients.

Intensive oral prophylaxis does not alter the tongue microbiome in young patients with chronic kidney disease: longitudinal, randomized, controlled study.

Gingivitis is a common intraoral disease in patients with chronic kidney disease (CKD), which poses a particular interdisciplinary challenge. We aimed to determine the influence of an intensive oral prophylaxis program (OPP) compared to standard prevention measures on the tongue microbiome of young patients with CKD. Thirty patients with CKD (mean age 14.2 ± 5.2 years) and generalized gingivitis were included. The effects of the intensive OPP were compared with standard prophylaxis according to statutory health insurance (treatment as usual, TAU) as a control. Tongue swabs were taken from the patients at baseline (t1) and after 3 (t2) and 6 (t3) months. Next-generation sequencing of 16S rDNA genes was used to quantitatively characterize microbial communities. There were no differences in the abundance, richness, or diversity of the observed genera and species between the two study groups at baseline or after 3 or 6 months. Furthermore, no change in predefined gingivitis and oral health bacterial clusters were found. At the phylum level, Firmicutes were decreased after intervention in the TAU group (t2TAU 42.9 ± 7.1 to t3TAU 34.8 ± 4.7 (npairs=14), p=0.003; false discovery rate 0.02). The decrease of Firmicutes was not significant in the OPP group. Despite the intensity of dental prophylaxis and decreasing clinical signs of inflammation and decreasing plaque amount, no clinically relevant changes in the tongue microbiome were observed. Our results confirm the conserved and stable nature of the tongue microbiome, even in children with CKD.

Sequencing and multiple antimicrobial resistance of Pseudomonas fluorescens isolated from Nile tilapia fish in Egypt.

Pseudomonas fluorescens is one of the main causes of septicemic diseases among freshwater fish, causing severe economic losses and decreasing farm efficiency. Thus, this research was aimed to investigate the occurrence of P. fluorescens in Nile Tilapia (O. niloticus) fish in Egypt, gene sequencing of 16SrDNA gene, and antimicrobial susceptibility. P. fluorescens strains were detected in 32% (128\400) of apparently healthy (9%; 36\400) and diseased (23%; 92\400) Nile tilapia fish. The highest prevalence was observed in gills of fish, 31.3% followed by intestine 26.9%, liver 24.2%, and kidneys 17.6%. The PCR results for the 16SrDNA gene of P. fluorescens showed 16SrDNA gene in 30% of examined isolates. Moreover, Homogeny and a strong relationship between strains of P. fluorescens was confirmed using 16SrDNA sequences. Beside the responsibility of 16SrDNA gene on the virulence of P. fluorescens. The results of antimicrobial susceptibility tests revealed that all strains were resistant to piperacillin (100%), followed by ceftazidime (29.7%), and cefepime (25.8%). The strains of P. fluorescence were highly sensitive to cefotaxime (74.2%), followed by ceftriaxone and levofloxacin (70.3% each). Interestingly, 29.7% of strains of P. fluorescens were multiple antimicrobial-resistant (MAR).

First report of bacterial panicle blight (BPB) in Rice caused by Burkholderia gladioli in Bangladesh.

Bacterial panicle blight (BPB) has become one of the most destructive diseases of rice worldwide and Burkholderia gladioli and B. glumae are two major pathogens causing BPB (1). This disease causes several types of damage, most importantly grain spotting, rot, and panicle blight, which can result in yield losses of 75% or more (1,3). In recent years, symptoms including sheath rot, grain spotting, grain rot, and panicle blight have been observed in both inbred and hybrid rice varieties. These symptoms resemble those of BPB and cause cultivar-dependent yield losses. (3) also reported the same symptoms for BPB. To confirm the cause of the disease, 21 rice panicles (Haridhan, a local variety) with typical BPB symptoms were collected from a farmer's field in the region of Mymensingh, Bangladesh during the rainy season in mid-October, 2021. Due to the severity of the outbreak, the panicles became dark brown and produced chaffy grains; nearly 100% of the rice panicles in that field were severely infected. To identify the causal pathogen(s), 1g of rice grains from 20 plants with typical BPB symptoms were surface-sterilized by immersing them in 70% ethanol for a few seconds followed by sodium hypochlorite solution (3%) for 1min. The grains were then rinsed with sterilized distilled water three times. Surface-sterilized grains were then ground with a mortar and pestle; 5mL of sterile distilled water was added during grinding. The extracted suspension (20µL) was then either streaked or spread onto the selective medium (S-PG) (2). Bacterial colonies showing purple color on the S-PG medium were selected and purified as candidate pathogens. For molecular characterization, species specific primers targeting gyrB gene were used to perform PCR and resulted in 479bp as reported by (4). To verify further, the PCR products of 16SF & 16SR were amplified and sequenced partially producing around 1400bp (1) and five 16SF partial sequences were deposited into NCBI GenBank (OP108276 to OP108280). 16S rDNA and gyrB revealed almost 99% homology with Burkholderia gladioli (KU851248.1, MZ425424.1) and B. gladioli (AB220893, CP033430) respectively using BLAST analysis. These purified bacterial isolates produced a diffusible light-yellow pigment on King's B medium indicating toxoflavin production (3). The candidate five bacterial isolates were then confirmed by inoculating 10ml suspension 108CFU/mL into the panicles and sheaths of BRRIdhan28 in net house condition as described previously (1). All of the bacterial isolates obtained from the spotted rice grains produced light brown lesions on the inoculated leaf sheath as well as spotting on the grain. To fulfill Koch's postulates, the bacteria were re-isolated from the symptomatic panicles and were confirmed as B. gladioli by analyzing the sequences of gyrB and 16s rDNA genes. Taken together, these results confirmed that B. gladioli is responsible for causing BPB in the rice grain samples that we collected. To our knowledge, this is the first report of BPB caused by B. gladioli in Bangladesh and further research is necessary to develop an effective disease management technique, or else the production of rice will be severely hampered.

First Report of Bark Split Disease Caused by Pectobacterium carotovorum on Artocarpus heterophyllus (Jackfruit) in China.

Jackfruit (Artocarpus heterophyllus) is widely cultivated in the tropical areas in the world. Jackfruit bark split disease occurred in the large-scale plantations of 18 cities and counties surveyed in Hainan since 2021, among which the incidence rate of serious orchards reached about 70%, and the mortality rate reached about 35%. Jackfruit bark split disease mainly harms tree branches and trunks, manifested as water stains, bark gumming, bark depression, bark cracking, and ultimately plant death. To identify the pathogen, Four samples with jackfruit bark split disease symptoms were collected, sterilized with 75% ethanol for 30 s, then soaked in 2% sodium hypochlorite (NaClO) for 5 mins, and finally rinsed continuously with sterilized distilled water. The sterilized tissues were placed on LB agar medium and incubated in illumination incubator at 28 ℃. Four milky white, round with neat edges, convex and smooth, translucent colonies were obtained. All isolates (JLPs-1 to JLPs-4) were Gram-negative, negative for oxidase, catalase and gelatin liquefaction. Amplification and sequencing of 16S rDNA gene from 4 isolates were conducted with the universal primers 27f /1492r (Lane et al. 1991). The BLASTn analysis of obtained JLPs-1 and JLPs-3 sequences (GenBank accession nos. OP942452 and OP942453) showed an identity percentage of 98.99% and 98.93% with Pectobacterium sp. (CP104733), respectively. Phylogenetic analysis based on 16S rDNA gene using the neighbor-joining method with MEGA 7.0 software revealed that JLPs-1 and JLPs-3 were clustered together with P. carotovorum reference strains. The four housekeeping genes gyrA, recA, rpoA and rpoS were partially sequenced for JLPs-1 isolates using primers gyrA1/gyrA4, recA1/recA2c, rpoS1/rpoS2 and rpoA F1/rpoA R1 (Loc et al. 2022), respectively. Multilocus sequence analyses identified the isolates from jackfruit as P. carotovorum. To further confirm the identification of Pectobacterium carotovorum, pelY gene, P. carotovorum subsp. Brasiliensis 16S-23S intergenic region (Pcb IGS) and P. carotovorum subsp. carotovorum (Pcc) specific fragment were amplified with primers Y1/Y2 (Darrasse et al. 1994), BR1f/L1r (Duarte et al. 2004) and EXPCCF/EXPCCR (Kang et al. 2003), respectively. A 540 bp target fragment was successfully amplified from JTPs only by EXPCCF/EXPCCR and there no bands for the other two primers. Pathogenicity test was performed in the field, and all the inoculated trees were 2-3-year-old 'Qiong Yin No.1' variety. Dense small holes were pierced with sterilized inoculation needle on four healthy jackfruit trees. Then punctured wounds were spraying-inoculated with bacteria suspension of JLPs-1 (108 CFU/ml), and finally wrapped with plastic wrap to moisturize. Two trees inoculated with sterile distilled water served as negative control. Typical symptoms of bark gumming, bark depression, bark cracking were observed on all of the inoculated trees at 17 dpi which just similar to those originally caused by P. carotovorum in the field, whereas negative control trees remained asymptomatic. The strains were re-isolated successfully from symptomatic jackfruit trees and were consistent with the biological and molecular biological characteristics of original strains, confirming that the pathogen of jackfruit bark split disease was Pectobacterium carotovorum. To our knowledge, this is the first report of P. carotovorum causing bark split disease on jackfruit in China.

A potential marker of radiation based on 16S rDNA in the rat model: Intestinal flora.

The gastrointestinal microbiota plays an important role in the function of the host intestine. However, little is currently known about the effects of irradiation on the microorganisms colonizing the mucosal surfaces of the gastrointestinal tract. The aim of this study was to investigate the effects of X-ray irradiation on the compositions of the large intestinal Microbiotas of the rat. The gut microbiotas in control mice and mice receiving irradiation with different dose treatment were characterized by high-throughput sequencing of the bacterial 16S rDNA gene and their metabolites were detected by gas chromatography-mass spectrometry. Unexpectedly, the diversity was increased mildly at 2Gy irradiation, and dose dependent decreased at 4Gy, 6Gy, 8Gy irradiation. The phyla with large changes in phylum level are Firmicutes, Bacteroides and Proteobacteria; the abundance ratio of Firmicutes/Bacteroides is inverted; and when 8Gy is irradiated, the phylum abundance level was significantly increased. At the genus level, the abundance levels of Phascolarctobacterium, Ruminococcaceae and Lachnospiraceae increased at 2Gy irradiation, and significantly decreased at 4Gy, 6Gy, and 8Gy irradiation; the abundance level of Prevotellaceae diminished at 2Gy irradiation, and enhanced at 4Gy, 6Gy, 8Gy irradiation; The abundance level of Violet bacteria (Christenellaceae) and Lactobacillus attenuated in a dose-dependent manner; Lachnoclostridium enhanced in a dose-dependent manner; Bacteroides was in 4Gy, 6Gy, 8Gy The abundance level increased significantly during irradiation; the abundance level of Shigella (Escherichia-Shigella) only increased significantly during 8Gy irradiation. Lefse predicts that the biomarker at 0Gy group is Veillonellaceae, the biomarker at 2Gy group is Firmicutes, the biomarkers at 4Gy group are Dehalobacterium and Dehalobacteriaceae, the biomarkers at 6Gy group are Odoribacter, and the biomarkers at 8Gy group are Anaerotruncus, Holdemania, Proteus, Bilophila, Desufovibrionales and Deltaproteobacteria. Overall, the data presented here reveal that X-ray irradiation can cause imbalance of the intestinal flora in rats; different doses of irradiation can cause different types of bacteria change. Representative bacteria can be selected as biomarkers for radiation damage and repair.This may contribute to the development of radiation resistance in the future.

Gut microbiota of cattle and horses and their use in the production of ethanol and lactic acid from timothy hay

The gut of herbivorous animals is an untapped source of the microbial consortium that could be used to improve the hydrolysis of lignocellulosic biomass. This study investigated the hydrolysis of Timothy hay by the anaerobic incubation with fecal inocula from four cows (CB) and four horses (HB) for 72 h. The microbial communities colonized timothy hay were analyzed using Illumina Mi-Seq sequencing of 16S rDNA genes. The source of the inoculum affected the structure of microbial communities that were dominated by phylum Firmicutes, Bacteroidetes, and Proteobacteria, and the dominant genera were Enterococcus, Streptococcus, Escherichia-Shigella, Bacteroides, and Prevotella. Moreover, several bacteria were observed exclusively in a specific group. For example, group CB had phylum Fibrobacterota and Spirochaetota besides genera Prevotellaceae YAB2003 and Bacteroides; and group HB had genera Weissella and Lactococcus. Moreover, function prediction revealed that the CB group has a higher abundance of genes related to carbohydrate metabolism. Group CB showed higher neutral detergent fiber degradability (NDFD), gas production, cellulase, xylanase, and xylose, and group HB showed higher dry matter degradability (DMD), glucose, ethanol, and lactic acid. The feces of cattle and horses represent a source of the bacterial consortium for enzyme production and the hydrolysis of lignocellulosic biomass.

Acute temperature stresses trigger liver transcriptome and microbial community remodeling in largemouth bass (Micropterus salmoides)

The largemouth bass is an important freshwater fish in China. As global climate changes, mass mortality in largemouth bass due to extremely low/high temperatures has aroused growing concern. In the present study, we investigated the physiological responses of the liver and intestine in largemouth bass to acute low/high-temperature stresses. Acute low/high-temperature stresses caused histological alterations such as structural damage in the liver. Based on liver transcriptomic data, a total of 6678 differentially expressed genes (DEGs) were detected, of which 2106, 5235, 3 and 449 DEGs were identified in fish treated with temperatures of 9 °C (serve cold-stress group, SC group), 18 °C (light cold-stress group, LC group), 30 °C (light heat-stress group, LH group), and 33 °C (serve heat-stress group, SH group), respectively. Further functional analysis found that the apoptosis, amino acid metabolism and lipid metabolism related signaling pathways were significantly enriched in fish after low-temperature stress, and protein processing in endoplasmic reticulum were significantly associated with heat stress. The weighted gene co-expression network analysis (WGCNA) results showed that multiple modules were significantly correlated with the LC or SC or SH groups, in which key signaling pathways were identified. Meanwhile, sequencing of the bacterial 16S rDNA gene demonstrated that acute high temperature stress reshaped the composition of the microbial community. Correlation analysis between bacteria and DEGs showed that bacteria could regulate pathways to affect the healthy status of host. This study provides the first systematic analysis about the adaptative responses of largemouth bass under acute temperature stresses by unveiling microbial, transcriptional, and histological changes, and identify microbial biomarkers/genes to monitor the health status of largemouth bass and to improve fish temperature tolerance, which will lay the foundation for further studies on the environmental stress response in largemouth bass.

In vitro evaluation of the antibacterial effect of Brazilian red propolis ethanol extract in the prevention of periodontal disease in dogs

Dental plaque (DP) is found on the surface of teeth and comprises a community of microorganisms that form a structured biofilm. Bacteria present in DP are potential periodontal pathogens when there is an imbalance in the healthy oral environment, and are precursors of periodontal disease (PD). In dogs, the treatments, such as mechanical removal, are difficult and expensive to apply. Therefore, in order to seek new therapeutic alternatives to control dental plaque in dogs, Brazilian red propolis ethanol extract (RPEE) was tested to evaluate its antibacterial effect on bacteria isolated from DP of dogs without PD. DP was collected from the supragingival dental surfaces of 10 dogs. Bacterial isolates of DP were identified by PCR and sequencing of 16S rDNA gene. The RPEE was obtained using the ultrasound ethanol extraction technique, and the chemical composition was obtained by HPLC-DAD and UV-spectrophotometry. In total, 29 different bacteria belonging to five genera were identified. Formononetin, biochanin A, liquiritigenin and daidzein were the major constituents of the RPEE. The cytotoxic effect showed cell viability after 24 h above 50 % at all concentrations evaluated. The minimum inhibitory concentration was between 37.5 and 150.0 µg/mL for all bacterial isolates. The minimal bactericidal concentration was between 150 and 1200 µg/mL for Gram-positive and 300–1200 µg/mL for Gram-negative bacteria. The results are promising and suggest that RPEE has significant antibacterial potential against the bacteria present in the DP of healthy dogs. Although further studies are still needed, the results suggest RPEE might be safely used in the prevention of periodontal disease.

Mixed pulmonary infection with four isolates of nontuberculous mycobacteria: a case report of mycobacterium bacteremicum infection

BackgroundA mixed pulmonary infection of Mycobacterium bacteremicum and three different isolates of nontuberculous mycobacteria (NTM) is an unusual clinical manifestation and have not yet been indicated. In this case report, we reported four isolates of NTM using phenotypic and genotypic test of pulmonary sample in Tehran, Iran.Case presentationWe report a case of severe pulmonary disease in a 19-year-old male patient with productive cough, shortness of breath, and low-grade fever for several weeks. The C-reactive protein (CRP) level (80.2 mg/L) and erythrocyte sedimentation rate (ESR) (95 mm/h) were high. The computed tomographic scan indicated bronchiectasis, nodular opacities, consolidation, and cavitary lesions on both sides. The result of purified protein derivative (PPD) test was equal to 15 mm. The sequences of hsp65, rpoB, and 16S rDNA genes indicated more than 99% homology to four isolates of nontuberculous mycobacteria (NTM), including Mycobacterium fortuitum, M. chelonae, M. mucogenicum, and M. bacteremicum. We found that all four strains were susceptible to amikacin, cefoxitin, ciprofloxacin, clarithromycin, imipenem, and linezolid. The patient was treated with ciprofloxacin, clarithromycin, and amikacin, along with Montelukast, for five months.ConclusionWe report a case of severe pulmonary infection by four isolates of NTM. After treatment, the patient reported complete resolution of the signs and a weight gain of 5 kg; also, the CRP and ESR were normal. Nine months after the infection diagnosis, a new CT scan revealed further improvements.

Natural association of the entomopathogenic nematode Heterorhabditis indica (Rhabditida: Heterorhabditidae) from the Philippines with the non-symbiotic bacterium Ochrobactrum anthropi (Proteobacteria: Brucellaceae)

BackgroundEntomopathogenic nematodes of the genera Heterorhabditis and Steinernema are widely used as biocontrol agents against diverse insect pests. Their association with symbiont bacteria is found to be the primary cause of insect mortality. The present study reported a comprehensive characterization of a local Philippine EPN isolate using congruent morpho-taxometrical and molecular data and the associated bacterium in the infective dauer juveniles.ResultsUsing an insect-baiting technique, a nematode isolate labeled F2H was recovered from the sandy soils in the coastal area of southern Philippines. Based on morphology and morpho-taxometrics, the F2H isolate collected from Iligan City was identified as Heterorhabditis indica. Analysis of the ITS region of rDNA revealed the highest sequence match to H. indica. The sequences of the bacterial 16S rDNA gene showed that the symbiont of H. indica presented 100% similitude with the sequences of Ochrobactrum anthropi. Further, pairwise alignment and phylogenetic analysis demonstrated that O. anthropi, O. lupini and O. cytisi shared 100% similarity and form a monophyletic clade. These strains have not presented any differences in the phylogenetic and 16S rDNA data, and O. lupini and O. cytisi should be considered a later heterotypic synonym of O. anthropi.ConclusionsThe description of H. indica-O. anthropi association was the first report in the Philippines. This provides additional account and collection of naturally isolated EPN-bacteria, contributing to limited knowledge on its diversity in the Philippines, which can be utilized in the biocontrol of pests after further comprehensive assessments.

Inactivated Whole Vaccine Inhibits Lethal Vibrio harveyi Infection in Oplegnathus punctatus

Aquaculture plays a key role in food production globally and provides a valuable source of protein and nutrition, addressing a worldwide growing demand. Oplegnathus punctatus (spotted knifejaw) is an economically important fish species with a high market value and demand. Previous studies on O. punctatus focused mainly on gonadal development, chromosomal microstructure, selective breeding, characterization of immune genes, and viral diseases. There is no published scientific research regarding vibriosis in this fish species. In this study, two potential pathogenic bacteria, Vibrio harveyi and Enterococcus gallinarum, were isolated from moribund cultured O. punctatus. The sequence of the universal 16S rDNA gene was used to identify potential pathogenic bacteria isolated from the moribund O. punctatus, and morphological assessments and API20E tests of the bacterial isolates were conducted to verify the identity and biochemical characteristics of the isolates. Injection of E. gallinarum did not lead to mortality in O. punctatus during the 21 days of observation. In contrast, fish died overnight when challenged with V. harveyi at 1.25 × 105 CFU/g body weight, suggesting that the cause of death of the cultured O. punctatus was V. harveyi infection. Antimicrobial sensitivity analyses revealed that the V. harveyi strain NTOU is sensitive to flumequine, doxycycline, oxolinic acid, and amoxycillin. Importantly, we demonstrated for the first time that intraperitoneal administration of an inactivated V. harveyi whole-cell vaccine resulted in a high level of protection against V. harveyi infection in O. punctatus.

Bacteria Community Vertical Distribution and Its Response Characteristics to Waste Degradation Degree in a Closed Landfill

The diversity, community structure and vertical distribution characteristics of bacteria in the surface and subsurface soil and water samples of a closed landfill in Shanghai Jiading District were investigated to reveal the relationships between natural waste degradation degree and the succession of bacterial community composition. High-throughput sequencing of bacteria 16S rDNA genes was used to analyze the bacterial community structure and diversity. The results showed that the diversity of bacteria in the surface samples was higher than that in the deep samples. Proteobacteria was the dominant phylum in all the samples, and the percentage increased with depth. At the genus level, Thiobacillus, Pseudomonas, Aquabacterium, and Hydrogenophaga were the dominant genera in surface, medium, deep and ultra-deep soils, respectively. The Bray–Curtis dissimilarity of the soil bacterial communities in the same layer was small, indicating that the community composition of the samples in the same layer was similar. The RDA result showed that ammonium, nitrate, pH and C/N significantly influenced the community structure of soil bacteria. This is of great relevance to understand the effect of natural waste degradation on bacterial communities in closed landfills.

Short Communication: Molecular identification and assessment of bacterial contamination of frozen local and imported meat and chicken in Basrah, Iraq using 16S rDNA gene

Abstract. Mohammad AJ, Alyousif NA. 2022. Short Communication: Molecular identification and assessment of bacterial contamination of frozen local and imported meat and chicken in Basrah, Iraq using 16S rDNA gene. Biodiversitas 23: 1598-1604. Meat is the main food source of daily meals with highly beneficial effects on human health. It is susceptible to the growth and reproduction of various bacteria due to its composition, which leads to its spoilage, and being an important carrier of pathogenic bacteria. The study aimed to isolate and identify the bacteria contamination of frozen local and imported meat and chicken samples using 16S rDNA gene. Twenty meat samples were collected randomly from local and imported meat sold in markets of Basrah province, Iraq. The results showed that the total aerobic bacteria count was within acceptable limits in all samples. Fifty-seven bacterial isolates were isolated and identified by amplification and sequencing of 16S rDNA gene, where the gram-positive bacteria were the most abundant 34(59.6%), and S. pasteuri is the most common species in the samples with 14 (24%) bacterial isolates out of the total isolates. Fifteen isolates were identified as new strains and their sequences were deposited at GenBank (NCBI) under different accession numbers (MZ964582-MZ964596). The meat and chicken have many bacteria on their surface, therefore the products must be frozen, and sanitary precautions should be taken into consideration.

Distribution, occurrence and molecular characterization of Bacillus related species isolated from different soil in Basrah Province, Iraq

Abstract. Alyousif NA. 2021. Distribution, occurrence and molecular characterization of Bacillus related species isolated from different soil in Basrah Province, Iraq. Biodiversitas 23: 679-686. Bacillus related species are widely predominant in various environments such as soil with diverse environmental conditions. These bacteria are one of the most explored groups of bacteria in industrial biotechnology due to their enzymes. The present study was aimed to isolate, screen and determine the distribution of Bacillus related species from soil samples. The total Bacillus related species counts in one gram of each soil sample ranged between 3.2 X 103 cfu/g and 4 X 103 cfu/g. A total of 43 isolates with different morphologies were isolated from the soil samples out of ten different habitats and the number of morphotypes recovered from each habitat varied from 2 to 7. The molecular identification of isolates by PCR technique and sequencing of 16S rDNA gene indicated that 43 isolates belonged to the genera Bacillus, Cytobacillus, Priestia and Peribacillus. The species Bacillus licheniformis and Bacillus subtilis were the most distributed species in the habitats. The bacterial isolates were showed 100% similarity, except 8 isolates were showed 99% similarity were identified as new strains and their sequences were deposited at the National Center for Biotechnical Information (NCBI). The phylogenetic tree was constructed by MEGA X software based on 16S rDNA gene sequences of Bacillus related species to evaluate their close relationship and evolution. The study revealed the higher diversity and distribution of Bacillus related species in the soil samples and the molecular technique was the best method for identifying Bacillus related species.

Biological Trace Information Extracted from Bioaerosols Using NGS Analysis

Bioaerosols are atmospheric particles with a biological trace, such as viruses, bacteria, fungi, and plant material such as pollen and plant debris. In this study, we analyzed the biological information in bioaerosols using next generation sequencing of the trace DNA. The samples were collected using an Andersen air sampler and separated into two groups according to particulate matter (PM) size: small (PM2.5) and large (PM10). Amplification and sequencing of the bacterial 16S rDNA gene, prokaryotic internal transcribed spacer 1 (ITS1) region and DNA sequence of a plant chloroplast gene (rbcL) were carried out using several sets of specific primers targeting animal and plant sequences. Lots of bacterial information was detected from the bioaerosols. The most abundant bacteria in several samples were of the Actinobacteria (class), Alphaproteobacteria, Bacilli, and Clostridia. For the animal detection using internal transcribed spacer 1, only uncultured fungi were detected in more than half of the hits, with a high number of Cladosporium sp. in the samples. For the plant identification, the ITS1 information only matched fungal species. However, targeting of the rbcL region revealed diverse plant information, such as Medicago papillosa. In conclusion, traces of bacteria, fungi, and plants could be detected in the bioaerosols, but not of animals using our primers.

Bacterial diversity dynamics in microbial consortia selected for lignin utilization

Lignin is nature’s largest source of phenolic compounds. Its recalcitrance to enzymatic conversion is still a limiting step to increase the value of lignin. Although bacteria are able to degrade lignin in nature, most studies have focused on lignin degradation by fungi. To understand which bacteria are able to use lignin as the sole carbon source, natural selection over time was used to obtain enriched microbial consortia over a 12-week period. The source of microorganisms to establish these microbial consortia were commercial and backyard compost soils. Cultivation occurred at two different temperatures, 30°C and 37°C, in defined culture media containing either Kraft lignin or alkaline-extracted lignin as carbon source. iTag DNA sequencing of bacterial 16S rDNA gene was performed for each of the consortia at six timepoints (passages). The initial bacterial richness and diversity of backyard compost soil consortia was greater than that of commercial soil consortia, and both parameters decreased after the enrichment protocol, corroborating that selection was occurring. Bacterial consortia composition tended to stabilize from the fourth passage on. After the enrichment protocol, Firmicutes phylum bacteria were predominant when lignin extracted by alkaline method was used as a carbon source, whereas Proteobacteria were predominant when Kraft lignin was used. Bray-Curtis dissimilarity calculations at genus level, visualized using NMDS plots, showed that the type of lignin used as a carbon source contributed more to differentiate the bacterial consortia than the variable temperature. The main known bacterial genera selected to use lignin as a carbon source were Altererythrobacter, Aminobacter, Bacillus, Burkholderia, Lysinibacillus, Microvirga, Mycobacterium, Ochrobactrum, Paenibacillus, Pseudomonas, Pseudoxanthomonas, Rhizobiales and Sphingobium. These selected bacterial genera can be of particular interest for studying lignin degradation and utilization, as well as for lignin-related biotechnology applications.

Descriptive Study of Gut Microbiota in Infected and Colonized Subjects by Clostridiodes difficile.

Clostridiodes difficile can lead to a range of situations from the absence of symptoms (colonization) to severe diarrhea (infection). Disruption of gut microbiota provides an ideal environment for infection to occur. Comparison of gut microbiota of infected and colonized subjects could provide relevant information on susceptible groups or protectors to the development of infection, since the presence of certain genera could be related to the inhibition of transition from a state of colonization to infection. Through high-throughput sequencing of 16S rDNA gene, we performed alpha and beta diversity and composition studies on 15 infected patients (Group CDI), 15 colonized subjects (Group P), and 15 healthy controls (Group CTLR). A loss of alpha diversity and richness and a different structure have been evidenced in the CDI and P groups with respect to the CTRL group, but without significant differences between the first two. In CDI and P groups, there was a strong decrease in phylum Firmicutes and an expansion of potential pathogens. Likewise, there was a loss of inhibitory genus of C. difficile germination in infected patients that were partially conserved in colonized subjects. Therefore, infected and colonized subjects presented a gut microbiota that was completely different from that of healthy controls, although similar to each other. It is in composition where we found that colonized subjects, especially in minority genera, presented differences with respect to those infected.