Identification of microbial agents in culture-negative brain abscess samples by 16S/18S rRNA gene PCR and sequencing

@#Despite clinical suspicion of an infection, brain abscess samples are often culture-negative in routine microbiological testing. Direct PCR of such samples enables the identification of microbes that may be fastidious, non-viable, or unculturable. Brain abscess samples (n = 217) from neurosurgical patients were subjected to broad range 16S rRNA gene PCR and sequencing for bacteria. All these samples and seven formalin-fixed paraffin-embedded tissue (FFPE) samples were subjected to species-specific 18S rRNA PCR for neurotropic free-living amoeba that harbour pathogenic bacteria. The concordance between smear and/or culture and PCR was 69%. One-third of the samples were smear- and culture-negative for bacterial agents. However, 88% of these culture-negative samples showed the presence of bacterial 16S rRNA by PCR. Sanger sequencing of 27 selected samples showed anaerobic/fastidious gram negative bacteria (GNB, 38%), facultative Streptococci (35%), and aerobic GNB (27%). Targeted metagenomics sequencing of three samples showed multiple bacterial species, including anaerobic and non-culturable bacteria. One FFPE tissue revealed the presence of Acanthamoeba 18S rRNA. None of the frozen brain abscess samples tested was positive for 18S rRNA of Acanthamoeba or Balamuthia mandrillaris. The microbial 16/18S rRNA PCR and sequencing outperformed culture in detecting anaerobes, facultative Streptococci and FLA in brain abscess samples. Genetic analyses of 16S/18S sequences, either through Sanger or metagenomic sequencing, will be an essential diagnostic technology to be included for diagnosing culture-negative brain abscess samples. Characterizing the microbiome of culture-negative brain abscess samples by molecular methods could enable detection and/or treatment of the source of infection.

Genetic analysis of mitochondrial pcox1 and ribosomal 18S rRNA genes in Eurytrema pancreaticum isolates from goats in Huaihua City, Hunan Province / 中国血吸虫病防治杂志

Objective To investigate the genetic variation of Eurytrema pancreaticum isolated from goats in Huaihua City, Hunan Province. Methods The partial sequence of mitochondrial cytochrome I (pcox1) and ribosomal 18S rRNA genes were amplified using a PCR assay in E. pancreaticum isolates from goats in Huaihua City, Hunan Province, and the PCR amplification products were sequenced. Then, the gene sequences were subjected to genetic variation and phylogenetic analyses. Results The sequences of the pcox1 and 18S rRNA genes were 430 bp and 1 857 bp in length in 18 E. pancreaticum isolates from goats in Huaihua City, Hunan Province, and there were 14 and 35 variation sites in pcox1 and 18S rRNA gene sequences, with intra-species genetic variations of 0 to 1.4% and 0 to 0.8%, respectively. The sequences of pcox1 and 18S rRNA genes had 99.0% to 99.8% and 99.5% to 99.8% homologies with those from E. pancreaticum Chinese strain recorded in the GenBank database. Consistent phylogenetic analysis results were found based on pcox1 and 18S rRNA genes. The 18 E. pancreaticum isolates from goats in Huaihua City were clustered into a clade with the known E. pancreaticum isolates registered in GenBank, and the clade with these 18 E. pancreaticum isolates was close to the clades with Eurytrema species and far from the clades with other trematodes. Conclusions The E. pancreaticum isolates from goats have a low genetic variation in Huaihua City, Hunan Province. Mitochondrial pcox1 and ribosomal 18S rRNA genes may serve as molecular markers for the studies on the genetic variation in goat-derived E. pancreaticum.

DNA barcoding of 4 species of cheyletid mites based on COI and 18S rRNA gene sequences / 中国血吸虫病防治杂志

Objective To analyze the sequences of mitochondrial cytochrome C oxidase subunit I gene (COI) and 18S ribosomal RNA gene (18S rRNA), so as to identify the feasible DNA barcodes for 4 species of cheyletid mites and improve the DNA barcoding database for cheyletid mites. Methods Cheyletid mite samples were collected from small-scale flour mills in Fuyang, Wuhu and Tongling cities of Anhui Province from May 2018 to July 2019, extracted and morphologically identified. Then, genomic DNA was extracted from a single cheyletid mite, and the COI and 18S rRNA gene sequences were obtained by PCR amplification, cloning and sequencing. The obtained sequences were aligned using the BLAST software. Multiple sequence alignment was done using the software ClustalX version 1.83 using the known gene sequences from cheyletid mites. The genetic distance was calculated using the software MEGA X, and the phylogenetic tree was created using the maximum likelihood method. Results The DNA barcoding results of Cheyletus malaccensis, C. carnifex and Cheletomorpha lepidopterorum were consistent with the morphological identification, while no sequences pertaining to Eucheyletia reticulate were retrieved in the GenBank database. The proportions of A + T were 69.6% and 55.1% in the COI and 18S rRNA sequences of 4 cheyletid mites species, respectively, and the numbers of base substitutions were 137 and 46, respectively. There were 154 to 321 and 58 to 99 inter-species variation loci in the COI and 18S rRNA gene sequences of 4 cheyletid mites species, respectively, and the intra-species genetic distance was all 0.020 or less in the COI and 18S rRNA gene sequences of 4 cheyletid mites species, with inter-species genetic distance of 0.235 to 0.583 and 0.078 to 0.114, respectively. Phylogenetic analysis based on COI and 18S rRNA genes showed that all four species of cheyletid mites were clustered into a branch with a 100% supportive rate, which was consistent with the morphological identification. Conclusion Mitochondrial COI gene is superior to 18S rRNA gene as DNA barcodes for 4 species of cheyletid mites, which is more suitable to be used to investigate the phylogenetic relationship of at genus and species levels.

Molecular authentication of Sarcandra glabra and its adulterants using three DNA molecular markers / 中草药

Objective: To identify Sarcandra glabra and its adulterants using three DNA molecular markers including 18 S rRNA gene, ITS2 sequence and SCAR marker, and then provide the basis for its molecular authentication. Methods: 18 S rRNA gene sequence of S. glabra was obtained by PCR amplification, cloning and sequencing, and then Blast comparison was made in NCBI. The ITS2 sequence of S. glabra was obtained by PCR amplification, sequencing and annotation in ITS2 Database. In the meanwhile, the ITS2 sequences of adulterants and other plants were collected from GenBank. Using MEGA5.5, the genetic distance was calculated between species and then the ITS2 sequences were aligned to construct a phylogenetic clustering tree. SCAR molecular marker of S. glabra was obtained by RAPD. After cloning and sequencing, specific primers were designed to amplify S. glabra and its adulterants. Results: The length of 18 S rRNA obtained in our research was 1 820 bp. Blast comparison revealed that there was 99% homology between S. glabra and Chloranthaceae, which proved to be 18 S rRNA gene of S. glabra. The length of the ITS2 sequence in our research was 500 bp. Genetic distance between S.glabra and its adulterants ranged from 0.190 to 0.219, which was far more than genetic distance among adulterants (0.000-0.074). Cluster analysis showed that S. glabra and its adulterants respectively clustered into a different branch, which was far away from other plants. In our research, we obtained SCAR molecular marker of S. glabra and then a pair of specific primers were designed. Using the pair of specific primers, specific products were amplified from genomic DNA of S. glabra, but no specific products were obtained from that of its adulterants. Conclusion: We could authenticate S. glabra and its adulterants effectively with the combination of three molecular markers for establishing a novel method to identify S. glabra and its adulterants, which provides a new idea for the authentication of S. glabra.

Cyclospora cayetanensis infections among diarrheal outpatients in Shanghai: a retrospective case study / 医学前沿

Cyclospora cayetanensis is a foodborne and waterborne pathogen that causes endemic and epidemic human diarrhea worldwide. A few epidemiological studies regarding C. cayetanensis infections in China have been conducted. During 2013, a total of 291 stool specimens were collected from patients with diarrhea at a hospital in urban Shanghai. C. cayetanensis was not detected in any of the stool specimens by traditional microscopy, whereas five stool specimens (1.72%, 5/291) were positive by PCR. These positive cases confirmed by molecular technology were all in the adult group (mean age 27.8 years; 2.94%, 5/170) with watery diarrhea. Marked infection occurred in the rainy season of May and July. Sequence and phylogenetic analyses of the partial 18S rRNA genes of C. cayetanensis isolated showed intra-species diversity of this parasite. This study showed, for the first time, that C. cayetanensis is a pathogen in outpatients with diarrhea in Shanghai, albeit at a low level. However, the transmission dynamics of this parasite in these patients remain uncertain.

Epidemic characteristics of thelaziasis and specie identification of Thelazia callipaeda by 18S rRNA gene / 中华地方病学杂志

Objective To study epidemiological characteristics of thelazjasis, in Zunyi and surrounding areas, morphology and 18S rRNA gene sequence of Thelazia callipaeda. Methods The Thelazia callipaeda was collected from several hospitals, including affiliated Hospital of Zunyi Medical College and Zunyi Aerospace Hospital, in Zunyi region since 2011 to 2015. Clinical data of infected human thelaziasis, including the patients' gender, age, residence and pets such as cats or dogs, were analyzed to find out the factors influencing the incidence. Morphology characteristics of female and male Thelazia callipaeda were observed under microscope. 18S rRNA gene of Zunyi Thelazia callipaeda was amplified by PCR, and the evolutional relationship was analyzed through the software MEGA 7.01 based on neighbour-joining (NJ) method. Homology was campared with 18S rRNA gene from GenBank in National Center of Biotechnology. Results Totally 25 cases had been reported during the study, of which 22 cases had more details. Based on the cases, we found the thelaziasis was increasing year by year. For instance, 2 cases (9.1%) were reported in 2011, 1 case (4.5%) in 2012, 3 cases in 2013 (13.6%), 10 cases (45.5%) in 2014 and 6 cases (27.3%) in 2015 . During the five years , totally 15 cases were treated between August and November, when the human thelaziasis was in typical epidemic peaks. We analyzed characteristics of the total cases reported to date. Most of the cases occurred in rural areas (20 cases). Majority of patients lived in rural region. And most cases were between 30 to 60 years old, indicating no age limit, especially, there were two cases who were at the ages of 8.5 months and 77 years old, respectively. Moreover, more women suffered from the disease than that in men, of which, the case number was 16 in women and 6 in men, and there were 7 cases who had cats or dogs at home. Under light microscope, the edge of Thelazia callipaeda had serrated cuticle with transverse striations. And male worm had a sharp peak at the tail end of Thelazia callipaeda, which cured to the ventrite and had two copulatory spicules, long one and short one, respectively. While female worm had a blunt tail, containing numerous eggs and rounded first-stage larvaes in a single line in the distal area of the uterus, near the vulva. Sequences of Thelazia callipaeda 18S rRNA gene from Zunyi and Oita Japan (AB538282.1) were showed homology of 100%, and they were clustered in a same branch of Phylogenetic tree. Conclusions Human thelaziasis cases in Zunyi region are increasing each year, and most of the cases have occurred in rural areas. 18S rRNA gene has a high homology with sequence AB538282.1 in Thelazia callipaeda. Combining clinical data, analysis of epidemiological characteristics and the characteristics of 18S rRNA should be good for specie identification and epidemiological analysis.

First molecular identification of Cryptosporidium by 18S rRNA in goats and association with farm management in Terengganu

Objective:To identify the prcvalencc of Cryptosporidium from goats in tlrec types of farm management systems in Terengganu,Malaysia and to determine the Crptosporidium species infecting goats by using 18S rRNA.Methods:A total of 478 fecal samples were randomly collected from goats in three farms;199 samples were collected from intensive farm,179 samples from semi-intensive farm and 1O0 samples from extensive farm.The samples were processed by using formolether concentration technique and stained by using modified Ziehl-Neelsen.Positive samples were performcd by using nested PCR analysis by using 18S rRNA.Results:Out of 478 goats,207 (43.3％) were found to be infected with Cryptosporidium.Goats reared under the intensive farm management system reported the highest prevalence of infection (49.7％),followed by intensive farm management system (41％) and the lowest prevalence was reported in the goats reared under semi-intensive management system (37.4％).Conclusions:The identified species found in goat was Cryptosporidium parvum.Future study on the zoonotic transmission of Cryptosporidium parvum in goats needs to be done in order to find the source of transmission of this parasite.

First molecular identification of Cryptosporidium by 18S rRNA in goats and association with farm management in Terengganu

Objective To identify the prevalence of Cryptosporidium from goats in three types of farm management systems in Terengganu, Malaysia and to determine the Cryptosporidium species infecting goats by using 18S rRNA. Methods A total of 478 fecal samples were randomly collected from goats in three farms; 199 samples were collected from intensive farm, 179 samples from semi-intensive farm and 100 samples from extensive farm. The samples were processed by using formol-ether concentration technique and stained by using modified Ziehl–Neelsen. Positive samples were performed by using nested PCR analysis by using 18S rRNA. Results Out of 478 goats, 207 (43.3%) were found to be infected with Cryptosporidium. Goats reared under the intensive farm management system reported the highest prevalence of infection (49.7%), followed by intensive farm management system (41%) and the lowest prevalence was reported in the goats reared under semi-intensive management system (37.4%). Conclusions The identified species found in goat was Cryptosporidium parvum. Future study on the zoonotic transmission of Cryptosporidium parvum in goats needs to be done in order to find the source of transmission of this parasite.

Isolation, morphological and molecular characterization of phytate-hydrolysing fungi by 18S rDNA sequence analysis

Phytate is the primary storage form of phosphate in plants. Monogastric animals like poultry, pigs and fishes have very low or no phytase activities in their digestive tracts therefore, are incapable to efficiently utilize phytate phosphorus from the feed. Phytase from microbial sources are supplemented to feedstuff of these to increase the uptake of phytate phosphorus. In the present work efforts were made to isolate and characterize proficient phytase producing fungi from soil. Phytase producing fungi were isolated using phytate specific medium. Fungal isolates were selected according to their higher phytase activities. These isolates were further characterized and identified by morphological and microscopic analysis and confirmed by amplification of 18S rRNA gene, using specific primers. This gene was subsequently sequenced and phylogenetic affiliations were assigned. Fungal isolates were identified as various species of Aspergillus. Phytases from these fungi could be utilized as a feed additive in poultry and swine industries.

Design of primer pairs for species-specific diagnosis of Leishmania (Leishmania) infantum chagasi using PCR / Construção de pares de primers para a detecção espécie-específica de Leishmania (Leishmania) infantum chagasi por PCR

The objective of this study was to design and evaluate new primers for species-specific detection of L. infantum chagasi using PCR. Two combinations of primer pairs were established with the aim of obtaining specific amplification products from the L. infantum chagasi 18S rRNA gene. The combinations of the primer pairs and the respective sizes of the PCR products, based on the U422465 GenBank reference sequence of L. infantum chagasi, were: LCS1/LCS3 (259 bp) and LCS2/LCS3 (820 bp). It was concluded that the new PCR assays optimized using the primer pairs LCS1/LCS3 and LCS2/LCS3 were effective for specific detection of L. infantum chagasi, with analytical sensitivity to detect 1 pg/µL of DNA.

Objetivou-se com este trabalho construir e avaliar novos primers para a detecção espécie-específicos de L. infantum chagasi por PCR. Foram estabelecidas duas combinações de pares de primers com a finalidade de obter produtos de amplificação específicos para o gene 18S rRNA de L. infantum chagasi. As combinações dos pares de primers e os respectivos tamanhos dos produtos de PCR, previstos conforme a sequência de referência utilizada (GenBank U422465) foram: LCS1/LCS3 (259 pb); LCS2/LCS3 (820 pb). Pôde-se concluir que os novos ensaios de PCR otimizados neste estudo, empregando os pares de primers LCS1/LCS3 e LCS2/LCS3, foram efetivos para a detecção específica de L. infantum chagasi, com sensibilidade analítica para detectar 1 pg/µL de DNA.

Sequencing of ribosomal 18S rRNA gene from Panax pseudoginseng var.notoginseng / 中草药

Object To identify the characteristics of 18S rRNA from the root of Panax pseudoginsengWall var. notoginseng (Burkill) Hoo et Tseng (PGSN). Methods The primers were designed according to the gene sequence of 18S rRNA from the model plant arabidopsis thaliana. 18S rRNA gene sequence of PGSN were cloned and sequenced and compared with that of the model plant A. thaliana and P. pseudoginseng subsp.Wall himalacus var. angustifolius. Results Part of the characteristics of ribosomal 18S rRNA gene sequence of PGSN from Jingxi, Guangxi Province were identified, which revealed that the 18S rRNA gene sequence of PGSN was 98% similar to that of P. pseudoginseng subsp. himalalaicus var . angustifolius and 96% similar to that of the model plant A. thaliana. Conclusion The use of informations obtained from the model plant, A. thaliana may promote the research progress of molecular biology of TCM drugs.

Application of gene technology in quality control of Chinese materia medica Ⅱ. Identification of Chinese Rhizoma Dioscoreae by DNA sequencing / 中草药

Object To distinguish the genuine Dioscorea polystachya Turcz. from other species of Dioscorea L. such as D. persimilis Prain et Burkill, D. japonica Thunb., and D. alata L., conventionally used in some localities by sequencing their nuclear ribosomal RNA subunit (18S rRNA), to provide mole cular evidences for the quality control of drugs of Dioscorea L. Methods By direct PCR sequencing to detect the homology of 18S rRNA sequence of different species of Dioscorea L.. Results The 18S rRNA gene sequence of genuine D. polystachya, D. persimilis and D. japonica showed identical length of 1810 bp, whereas that of D. alata was 1807 bp. Multiple sequence alignment of 18S rRNA gene showed that the homology was identical between D.persimilis and the genuine D. polystachya; but a difference of 99.89% with D. japonica and 97.51% with D. alata. Conclusion DNA sequencing can provide an accurate and reliable mean for the identification of the origin and genuineness of Dioscorea L..

Homology of 18S small subunit ribosomal RNA gene among species and strains of Schistosoma and sensitivity of PCR assay to detect single cercaria / 中国血吸虫病防治杂志

Objective To research the homology of 18S small subunit ribosomal RNA(18S-rRNA) gene about Chinese Mainland and Philippine strains of Schistosoma japonicum,and Schistosoma mansoni,and the possibility to establish the PCR assay based on the gene for detecting the cercaria in a low density level. Methods The genomic DNAs of Chinese Mainland and Philippine strains of S.japonicum,and S.mansoni were extracted. The PCR assay was used to detect the identical target DNA elements in the above genome team and the homology of their genes was compared. The single cercaria was respectively treated with the method of heating in boiling water,the method of treating with ammonia and the method of treating with NaOH,HCl and ethanol,and the single treated cercaria and the single cercaria without treating were used as the templates to amplify the target DNA by using the PCR assay,and the detection rates of the PCR assay to detect the single cercaria treated with the different methods were calculated and compared. Results With the genomic DNAs of Chinese Mainland and Philippine strains of S.japonicum and S.mansoni as the templates,the target DNA element of which sequence length was 469 bp was all amplified by PCR. The target DNA was all amplified by PCR to the single cercaria treated with ammonia and the method of treating with NaOH,HCl and ethanol. However,only 50 percent of specimens of the single cercaria without treating and the single cercaria treated with the method of heating in boiling water were amplified to the target DNA by PCR. Conclusions The 18S-rRNA gene has the general homology among the species and strains of Schistosoma. The sensitivity of the PCR assay to detect the low density cercaria treated with ammonia or the method of treating with NaOH,HCl and ethanol is higher than that of the single cercaria without treating or treated with the method of heating in boiling water.