OBJECTIVETo explore a new method of rapid and reliable diagnosis of bacterial infectious diseases such as purulent meningitis and septicemia.

METHODSA pair of universal primers and a set of probes (including universal fluorescence probe, Gram-positive probe and Gram-negative probe) were designed based on the bacterial highly conserved region of 16S rRNA gene. By using the FQ-PCR method, 12 standard strains, 23 clinical cultural isolations and the controls such as HBV, Cryptococcus histolyticus, Blastomyces albicans and human DNA were detected with the three kinds of probes. The correlation among the results of the three kinds of probes detection was analyzed.

RESULTSThe determination of 16S rRNA gene with FQ-PCR was a highly specific and sensitive method and not cross-reactive with human DNA, virus or fungi. The least amount of 10 copies of 16S rRNA gene which corresponded to 2 bacteria could be detected with FQ-PCR. Twelve standard strains and 23 clinical cultural isolations were detected by FQ-PCR with the three kinds of probes mentioned above. All samples presented positive results using the universal probe. The results of 16S rRNA gene detected by the Gramjpositive probe were positive to the 18 G+ strains. The results of 16S rRNA gene detected by the Gram-probe were positive to the 17 G- strains.

CONCLUSIONSThe FQ-PCR technique was established for bacteria quantifying and typing using the universal primer and the double type probes. This method was convenient and rapid in detecting, quantifying and typing bacteria, with a high specificity and sensitivity.

Fluorescence ; Genes, rRNA ; Humans ; Polymerase Chain Reaction ; methods ; RNA, Bacterial ; genetics ; RNA, Ribosomal, 16S ; genetics

Ma-Mu-Ran Antidiarrheal Capsules (MMRAC) is traditional Chinese medicine that has been used to treat diarrhea caused by acute enteritis (AE) and bacillary dysentery in Xinjiang (China) for many years. However, the potential therapeutic mechanism of MMRAC for AE and its regulatory mechanism on host metabolism is unclear. This study used fecal metabolomics profiling with GC/MS and 16S rRNA gene sequencing analysis to explore the potential regulatory mechanisms of MMRAC on a dextran sulfate sodium salt (DSS)-induced mouse model of AE. Fecal metabolomics-based analyses were performed to detect the differentially expressed metabolites and metabolic pathways. The 16S rRNA gene sequencing analysis was used to assess the altered gut microbes at the genus level and for functional prediction. Moreover, Pearson correlation analysis was used to integrate differentially expressed metabolites and altered bacterial genera. The results revealed that six intestinal bacteria and seven metabolites mediated metabolic disorders (i.e., metabolism of amino acid, carbohydrate, cofactors and vitamins, and lipid) in AE mice. Besides, ten altered microbes mediated the differential expression of eight metabolites and regulated these metabolisms after MMRAC administration. Overall, these findings demonstrate that AE is associated with metabolic disorders and microbial dysbiosis. Further, we present that MMRAC exerts protective effects against AE by improving host metabolism through the intestinal flora.
Animals ; Antidiarrheals/pharmacology* ; Capsules ; Enteritis/genetics* ; Feces/microbiology* ; Genes, rRNA ; Metabolomics ; Mice ; RNA, Ribosomal, 16S/genetics*

BACKGROUND: Osteoporosis (OP) has become a major public health issue, threatening the bone health of middle-aged and elderly people from all around the world. Changes in the gut microbiota (GM) are correlated with the maintenance of bone mass and bone quality. However, research results in this field remain highly controversial, and no systematic review or meta-analysis of the relationship between GM and OP has been conducted. This paper addresses this shortcoming, focusing on the difference in the GM abundance between OP patients and healthy controls based on previous 16S ribosomal RNA (rRNA) gene sequencing results, in order to provide new clinical reference information for future customized prevention and treatment options of OP. METHODS: According to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), we comprehensively searched the databases of PubMed, Web of Science, Embase, Cochrane Library, and China National Knowledge Infrastructure (CNKI). In addition, we applied the R programming language version 4.0.3 and Stata 15.1 software for data analysis. We also implemented the Newcastle-Ottawa Scale (NOS), funnel plot analysis, sensitivity analysis, Egger's test, and Begg's test to assess the risk of bias. RESULTS: This research ultimately considered 12 studies, which included the fecal GM data of 2033 people (604 with OP and 1429 healthy controls). In the included research papers, it was observed that the relative abundance of Lactobacillus and Ruminococcus increased in the OP group, while the relative abundance for Bacteroides of Bacteroidetes increased (except for Ireland). Meanwhile, Firmicutes, Blautia, Alistipes, Megamonas, and Anaerostipes showed reduced relative abundance in Chinese studies. In the linear discriminant analysis Effect Size (LEfSe) analysis, certain bacteria showed statistically significant results consistently across different studies. CONCLUSIONS: This observational meta-analysis revealed that changes in the GM were correlated with OP, and variations in some advantageous GM might involve regional differences.
Middle Aged ; Aged ; Humans ; Gastrointestinal Microbiome/genetics* ; RNA, Ribosomal, 16S/genetics* ; Genes, rRNA ; Osteoporosis ; Feces

Animals ; Animals, Wild ; microbiology ; Genes, rRNA ; RNA, Ribosomal, 16S ; genetics ; Rats ; Rickettsia ; classification ; genetics ; Rickettsia Infections ; microbiology ; veterinary

OBJECTIVETo investigate the drug-resistance of Acinetobacter baumannii (Ab) isolated from patients in burn ward, and study the incidence of 16S rRNA methylase genes mediated high-level aminoglycoside drug-resistance and its mechanism of transfer.

METHODSA total of 40 Ab clinical isolates were collected from burn ward in Gansu Province People's Hospital from May 2006 to Dec. 2007. The sensitivity of Ab for 20 antibiotics were determinated by K-B agar diffusion. The minimal inhibitory concentrations (MIC) of amikacin, gentamicin, tobramycin, netilmicin, isepamicin and kanamycin against Ab strains were determinated by agar dilution. Five kinds of 16S rRNA methylase genes including armA, rmtA, rmtB, rmtC, rmtD were amplified by PCR, the positive PCR-products were purified and sequenced, and the plasmid were extracted by alkaline lysis. The transferability of drug-resistance were determinated by conjugation and plasmid transformation tests.

RESULTSThe drug-resistance rates of Ab against six aminoglycosides antibiotics was 72.5%, 72.5%, 70.0%, 67.5%, 70.0%, 70.0%, respectively. Twenty five strains were resistant to six aminoglycosides antibiotics (62.5%), among which 10 isolates were armA-positive (40.0%); rmtA, rmtB, rmtC and rmtD-positive isolates were not found. Ten transformants and 10 conjugates showed high-level resistance against aminoglycosides antibiotics, all of which the value of MIC > or = 256 microg/mL carried armA gene.

CONCLUSIONSThe drug-resistance of Ab clinical isolates have high drug-resistance. 16S rRNA methylases gene exists in Ab and locates in plasmid chromosome.

Acinetobacter baumannii ; enzymology ; genetics ; isolation & purification ; Burn Units ; Burns ; microbiology ; Drug Resistance, Bacterial ; genetics ; Genes, Bacterial ; Genes, rRNA ; Humans ; Methyltransferases ; genetics ; Plasmids

There have been reports in Korea of imported malaria cases of four Plasmodium species, but there has been no report of imported Plasmodium ovale malaria confirmed by molecular biological methods. We report an imported case of that was confirmed by Wright-Giemsa-stained peripheral blood smear and nested polymerase chain reaction targeting the small subunit ribosomal RNA gene. The amplified DNA was sequenced and compared with other registered P. ovale isolates. The isolate in this study was a member of the classic type group. The patient was a 44-yr-old male who had worked as a woodcutter in Cote d'Ivoire in tropical West Africa. He was treated with hydroxychloroquine and primaquine and discharged following improvement. In conclusion, P. ovale should be considered as an etiology in the imported malaria in Korea, because the number of travelers to P. ovale endemic regions has recently increased.
Sequence Analysis, DNA ; Polymerase Chain Reaction ; Plasmodium ovale/*genetics ; Male ; Malaria/*diagnosis/etiology ; Humans ; Genes, rRNA ; Adult

Acanthamoeba, a free-living amoeba, is widely distributed in the environment, water sources, soil, dust, and air. It can cause keratitis in contact lens wearers with poor hygiene and also fatal granulomatous amebic encephalitis (GAE) in immunocompromised hosts. The aim of this study was to gain some insights into the distribution and genotypes of the potentially pathogenic species of Acanthamoeba present in water sources in north of Iran. Total 43 Acanthamoeba species were isolated from 77 water samples taken from different water sources within the Mazandaran province in Northern Iran (Sari city and suburbs). Isolates were identified based on cyst and trophozoite morphological characteristics as well genetics. PCR fragments corresponding to the small-subunit 18S rRNA gene were sequenced for 20 of 43 positive isolates. The results revealed that 83.3% of sequenced isolates belonged to the T4 genotype and the rest belonged to the T2 genotype. Our results indicated that Acanthamoeba is widely distributed in Sari city. As the incidence in Iran of amoebic keratitis has increased in recent years, the exact estimation of the prevalence of this amoeba and its predominant genotype may play a crucial role in prevention of the disease. Sari city has several rivers, seashores, and natural recreational amenities, which attract visitors during the year. This is the first report of Acanthamoeba genotypes from water sources in Sari city, Mazandaran province of Iran, and the results suggest that more attention is needed to protect the visiting population and immunocompromised individuals.
Acanthamoeba* ; Amoeba ; Dust ; Encephalitis ; Genes, rRNA ; Genetics ; Genotype ; Hygiene ; Immunocompromised Host ; Incidence ; Iran* ; Keratitis ; Parasites* ; Polymerase Chain Reaction ; Prevalence ; Rivers ; Soil ; Trophozoites ; Water

Echinostoma hortense (Digenea: Echinostomatidae) is one of the intestinal flukes with medical importance in humans. However, the mitochondrial (mt) genome of this fluke has not been known yet. The present study has determined the complete mt genome sequences of E. hortense and assessed the phylogenetic relationships with other digenean species for which the complete mt genome sequences are available in GenBank using concatenated amino acid sequences inferred from 12 protein-coding genes. The mt genome of E. hortense contained 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 non-coding region. The length of the mt genome of E. hortense was 14,994 bp, which was somewhat smaller than those of other trematode species. Phylogenetic analyses based on concatenated nucleotide sequence datasets for all 12 protein-coding genes using maximum parsimony (MP) method showed that E. hortense and Hypoderaeum conoideum gathered together, and they were closer to each other than to Fasciolidae and other echinostomatid trematodes. The availability of the complete mt genome sequences of E. hortense provides important genetic markers for diagnostics, population genetics, and evolutionary studies of digeneans.
Amino Acid Sequence ; Base Sequence ; Databases, Nucleic Acid ; Dataset ; Echinostoma* ; Echinostomatidae ; Fasciolidae ; Genes, rRNA ; Genetic Markers ; Genetics, Population ; Genome ; Genome, Mitochondrial* ; Humans ; RNA, Transfer ; Trematoda

Soil archaea and fungi play important roles in the greenhouse soil ecosystem. To develop and apply rich microbial resources in greenhouse ecological environment, and to understand the interaction between microbes and plants, we constructed archaeal 16S rRNA and fungal 18S rRNA gene libraries to analyze the compositions of archaeal and fungal communities. Total greenhouse soil DNA was directly extracted and purified by skiving-thawing-lysozyme-proteinase K-SDS hot treatment and treatment of cetyltriethylammnonium bromide (CTAB). After PCR amplification, retrieving, ligating, transforming, screening of white clones, archaeal 16S rRNA and fungal 18S rRNA gene libraries were constructed. The sequences of archaea and fungi were defined into operational taxonomic units (OTUs) when 97% similarity threshold for OTU assignment was performed by using the software DOTUR. Phylogenetic analysis showed that crenarchaeota and unidentified-archaea were the two major sub-groups and only a few of euryarchaeota existed in the archaeal clone library, total 45 OTUs. All the crenarchaeota belonged to thermoprotei; except for Basidiomycotina, the other four sub-group fungi were discovered in the fungal library, total 24 OTUs. The diversities of archaea were very abundant and a few euryarchaeota (methanebacteria) existed in the archaeal clone library, it might be directly related to the long-term high temperature, high humidity, and high content of organic matter. The limitation of oxygen was the other reason for causing this phenomenon; Ascomycotina (over 80%) was the dominant sub-groups in fungal library. It was because most of the plant fungal diseases belonged to soil-borne diseases which gone through the winter by the ways of scierotium or perithecium and became the sources of primary infection.
Archaea ; genetics ; growth & development ; Biodiversity ; Cloning, Molecular ; Cucumis sativus ; growth & development ; Fungi ; genetics ; growth & development ; Gene Library ; Genes, rRNA ; Microclimate ; Phylogeny ; Plant Roots ; microbiology ; RNA, Archaeal ; genetics ; RNA, Fungal ; genetics ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 18S ; genetics ; Soil Microbiology

OBJECTIVETo investigate the prevalence of Helicobacter pylori (H. pylori) resistance to clarithromycin (CLM) in children and to demonstrate the correlation of 23S rRNA gene mutation to clarithromycin resistance of Helicobacter pylori isolates.

METHODSTotally 108 clinical strains of H. pylori were isolated from gastric biopsy specimens obtained from children who underwent endoscopy during the period from October 2002 to January 2004 in Children's Hospital Affiliated to Medical College of Zhejiang University. H. pylori was identified by morphology and biochemical tests after culture. Clarithromycin susceptibility of H. pylori isolates was determined by both E-test and two-fold agar dilution method. A strain was considered resistant when the MIC was defined as >or= 1 microg/ml. Genome DNAs of the 108 isolates were extracted and prepared for PCR to detect the corresponding gene in the V domain of the 23S rRNA. The amplified fragments were recognized and analyzed by restriction fragment length polymorphism (RFLP) when an additional restriction site is created by the mutation. The PCR products of all sensitive and resistant strains were digested with restriction enzyme BbsI and BsaI and were analyzed on a 1.5% agarose gel to discriminate different kinds of mutant genotype.

RESULTSSixteen of 108 isolates of H. pylori were resistant to clarithromycin by the agar dilution method and E-test method in clinical isolates from children, and the CLM resistance rate was 14.8% (16/108) with MICs ranging from 1 microg/ml to 128 microg/ml. Comparison of results of the two methods showed that these two methods were quite consistent in determination of susceptibility and resistance. The target fragment 425 bp in length containing 23S rRNA corresponding gene was successfully amplified. An A2144G mutation digested with BsaI was detected in 13 resistant isolates, but an A2143G mutation digested with BbsI in only 3 among all 16 clarithromycin resistant strains. None of the sensitive isolates was cleaved by either BsaI or BbsI enzyme, indicating that there was no mutation on them. It was also found that all the fragments from the resistant strains were not completely digested, and 425 bp uncut fragments were also visible and showed three bands indicating that they were heterozygotic strains with a mixture of wild-types and A-->G genotypes. In addition, in this study, no statistically significant difference between mutations at positions 2143 and 2144 with respect to the MIC was observed (r = 0.035, P > 0.05).

CONCLUSIONA high prevalence of clarithromycin-resistant H. pylori strains were detected among strains isolated from Chinese children studied. The 23S rRNA gene mutation at positions A2143G and A2144G plays an important role in clarithromycin resistance of H. pylori and A2144G mutation is the predominant finding among the resistant strains.

Anti-Bacterial Agents ; pharmacology ; Biopsy ; Child ; Clarithromycin ; pharmacology ; Drug Resistance, Bacterial ; genetics ; Genes, rRNA ; Helicobacter Infections ; epidemiology ; genetics ; Helicobacter pylori ; drug effects ; genetics ; isolation & purification ; Humans ; Microbial Sensitivity Tests ; Mutation ; Prevalence ; Stomach ; microbiology ; pathology