Bacterial Genomic DNA Extraction Research Articles

Engineering the bacteriophage 80 alpha endolysin as a fast and ultrasensitive detection toolbox against Staphylococcus aureus

The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 102 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply.

Molecular detection of Brucella melitensis in sheep from district Budgam, Jammu and Kashmir

Farmers reported abortions and stillbirths in 600 sheep including 471 pregnant ewes. A complete investigation including physical examination of livestock, postmortem examination of stillbirths, serological examination of ewes and molecular study of abomasal contents, peritoneal fluid, liver and kidney tissue collected from still born lambs was done to ascertain the cause of mortality at Disease Investigation Laboratory, Nowshera, Srinagar. Also, on spot heat fixed tissue smears were prepared for Gram’s staining and accordingly observed for presence of organism. Extraction of bacterial genomic DNA from samples from still born lambs was performed and obtained DNA extracts were checked by agarose gel electrophoresis and stored at -20°C till further use. The PCR conducted on the abomasal, peritoneal contents and liver and kidney tissues revealed 731 bp band after the gel electrophoresis, confirming Brucella melitensis as cause of stillbirths in the flock. The investigation revealed that Brucella melitensis was cause of stillbirths and abortions in sheep flocks.

Cas14a1-advanced LAMP for ultrasensitive and visual Pathogen diagnostic

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas enzymes have been widely applied for biosensor development, combined with various isothermal amplification strategies (IAS) to boost sensitivity and specificity. Currently, the unstable assay and tedious manipulation usually hinder its practical applications. Here, a Cas14a1-advanced LAMP assay (CALA) combined with Rapid Extraction of Bacterial Genomic DNA (REBGD) is proposed for pathogen detection. For rapid CALA, a single stranded fluorescence reporter and ssDNA-gold nanoparticles (AuNPs) are used as signal indicators to establish ultrasensitive and visual platforms. This assay displays precise detection of bacteria, which can achieve an ultrasensitive limit of detection (LOD) 10 aM target genomic DNA. Furthermore, the high reliability of pathogen diagnostic for contrived samples is validated through the rapid visual CALA platform, demonstrating the promising practical testing availability of pathogen detection.

In vitro activities of antimicrobial combinations against planktonic and biofilm forms of Stenotrophomonas maltophilia.

To investigate the in vitro activity of antibiotic combinations against Stenotrophomonas maltophilia isolates and their associated biofilms. Thirty-two S. maltophilia clinical isolates with at least twenty-five different pulsotypes were tested. The antibacterial activity of various antibiotic combinations against seven randomly selected planktonic and biofilm-embedded S. maltophilia strains with strong biofilm formation was assessed using broth methods. Extraction of bacterial genomic DNA and PCR detection of antibiotic resistance and biofilm-related genes were also performed. The susceptibility rates of levofloxacin (LVX), fosfomycin (FOS), tigecycline (TGC) and sulfamethoxazole-trimethoprim (SXT) against 32 S. maltophilia isolates were 56.3, 71.9, 71.9 and 90.6%, respectively. Twenty-eight isolates were detected with strong biofilm formation. Antibiotic combinations, including aztreonam-clavulanic (ATM-CLA) with LVX, ceftazidime-avibactam (CZA) with LVX and SXT with TGC, exhibited potent inhibitory activity against these isolates with strong biofilm formation. The antibiotic resistance phenotype might not be fully caused by the common antibiotic-resistance or biofilm-formation gene. S. maltophilia remained resistant to most antibiotics, including LVX and β-lactam/β-lactamases; however, TGC, FOS and SXT still exhibited potent activity. Although all tested S. maltophilia isolates exhibited moderate-to-strong biofilm formation, combination therapies, especially ATM-CLA with LVX, CZA with LVX and SXT with TGC, exhibited a higher inhibitory activity for these isolates.

Vaginal carriage of Haemophilus influenzae in a non-pregnant reproductive-age population

BackgroundHaemophilus influenzae (Hi) is an emerging cause of early onset neonatal sepsis, but mechanisms of transmission are not well understood. We aimed to determine the prevalence of vaginal carriage of Hi in reproductive age women and to examine behavioral and demographic characteristics associated with its carriage.MethodsWe performed a secondary analysis of stored vaginal lavage specimens from a prospective cohort study of nonpregnant reproductive-age women. After extraction of bacterial genomic DNA, samples were tested for the presence of the gene encoding Haemophilus protein d (hpd) by quantitative real-time polymerase chain reaction (PCR) using validated primers and probe. PCR for the V3-V4 region of the 16 S rRNA gene (positive control) assessed sample quality. Samples with cycle threshold (CT) value < 35 were defined as positive. Sanger sequencing confirmed the presence of hpd. Behavioral and demographic characteristics associated with vaginal carriage of Hi were examined.Results415 samples were available. 315 (75.9%) had sufficient bacterial DNA and were included. 14 (4.4%) were positive for hpd. There were no demographic or behavioral differences between the women with Hi vaginal carriage and those without. There was no difference in history of bacterial vaginosis, vaginal microbiome community state type, or presence of Group B Streptococcus in women with and without vaginal carriage of Hi.ConclusionHi was present in vaginal lavage specimens of 4.4% of this cohort. Hi presence was unrelated to clinical or demographic characteristics, though the relatively small number of positive samples may have limited power to detect such differences.

Determination of toxicity and radioprotective properties of bacterial and fungal eumelanin pigments

Purpose Determination of the protective property of melanin, an organic polymer class consisting of phenolic and/or indolic compounds isolated from bacteria and fungi, against fast neutron radiation. To show that these melanin samples, which also have antioxidant and metal chelating properties, can be used as an active ingredient for a drug to be developed against neutrons used in nuclear research and medicine. Materials and methods Bacterial and fungal media were prepared, and melanin pigments were produced and isolated. For molecular characterization of pigments, bacterial genomic DNA extraction, 16S rDNA gene amplification processes, and fungal genomic DNA extraction, ITS1, and ITS4 Gene Regions amplification were performed. The DEL assay was implemented to determine the genotoxicity properties of bacterial and fungal melanin pigments. Samples were prepared in a pad measuring 10 ml volume (60 × 15 mm) at a concentration of 0.2–1 microgram in 1% agarose gel for radiation-absorbed dose measurements. Absorption measurements were made using 241Am-Be fast neutron source and Canberra brand NP series BF3 gaseous detector to determine the neutron radiation absorption capacity of all samples. The results obtained to determine the absorption degrees of melanin samples were compared with paraffin and normal concrete, which are widely used in neutron radiation shielding studies. Results Melanin pigments were obtained using different bacteria and fungi strains. Afterwards, the fast neutron radiation absorption capacity of these purified pigments were determined. Compared to reference samples, these pigments were found to have slightly lower radiation absorbing ability. In addition to these experiments, cytotoxicity tests were carried out using the Yeast DEL assay technique to evaluate the potential for use of these organic pigments in fields such as medicine and pharmacology. According to the results obtained from the tests, it was determined that these melanin samples did not have any toxic effects. Conclusion It was determined that these melanin samples have the potential to be used as a radioprotective drug active substance to protect the tissues and cells of people exposed to neutron radiation after a nuclear accident or nuclear war. Giving a drug that will be developed by using these active ingredients before or after people are exposed to a radiation environment can provide great benefits.

First report of bacterial soft rot caused by Enterobacter mori affecting host watermelon.

InJune2021,bacterial stem rot-likesymptomswere observed on the stems and leaves of watermelon (Citrullus lanatus cv. 'Zaojia') in Pingyu County, Zhumadian City, Henan Province, China (32.44N 114.24E), which showed brown to dark brown lesions on the stems (Fig. 1A). The stems then became scorched, and the leaves showed necrotic lesions with small water-soaked spots (Fig. 1B). Watermelon is a very important economic plant in this small county, where the watermelon planting area accounts for about 15% of the arable land area. Approximately 2 hectares of 'Zaojia' have been investigated, and the disease incidence rates were almost 20~30%, thus, causing severe economic losses. Ten symptomatic watermelon stems and leaves were randomly collected based on the typical symptoms, brought into the Lab and used to isolate the pathogen. Each infected tissue was excised and cut into small pieces (about 5 mm×5 mm) and surface disinfected with 1% NaClO for 3 min. The pieces were then rinsed three times in sterile distilled water (SDW) and dried by airing. These pieces (4-5 pieces per sample) were macerated in 200 μL SDW for 60s in a sterile mortar and pestle. A volume of 5 μL suspensions of each sample were streaked onto two LB agar plates and incubated for 48h at 28°C in the dark. After incubation, the colonies on LB agar plate were small, round, raised, white to cream-colored, and had smooth margins (Fig. 2). Two strains from each plate were selected. The genomic DNA of all 40 strains was extracted using a Bacterial Genomic DNA Extraction Kit D1600 (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer's instructions. The 16S ribosomal RNA gene (27F:5'-AGA GTT TGA TCC TGG CTC AG-3', /1492R: 5'-CTA CGG CTA CCT TGT TAC GA-3'), and the three housekeeping genes, including gyrB (Trantas et al., 2013), icdA and proA (Ma et al., 2007), were amplified. Sequence analysis showed that 40 strains shared the same sequence, so only one sequence was submitted into GenBanK.The 16s rDNA partial sequences (SUB12134746) shared 100% similarity with E.mori (CP084692.1), and the gyrB (OP676246), icdA (OP676248) and proA (OP676247) genes shared 98.67%, 99.39% and 97.99% homology with those of E. mori (CP084692.1), respectively. Besides, the phylogenetic tree analysis based on multi-housekeeping gene joint gryB-icdA-proA showed that E.mori（OP676246-OP676248- OP676247）from watermelon was culsterd with the E.mori (CP084692.1) from South Korea and E.mori (CP055276.1) from kiwifruit (Fig. 3). Thus, E.moriwas confirmed to be the pathogen responsible for bacterial soft rot of watermelon in this study. To confirm the pathogenicity, 15-day-old healthy cv. 'Zaojia' watermelon seedlings were inoculated by spraying all the seedlings with a bacterial suspension (1×10 8 CFU mL-1) at an incubation temperature of 28 °C and 70% relative humidity, and sterile distilled liquid LB medium was applied as a negative control treatment. Three times were conducted for the isolate, and each time included nine watermelon plants. After 10 days, only the inoculated cotyledons and leaves with the bacterial suspension showed bacterial leaf spots that resembled those observed on naturally infected watermelon cotyledons and leaves (Fig. 4A-C), whereas the control plants remained asymptomatic (Fig. 4D). Simultaneously, the watermelon stems were inoculated with the bacterium in vitro. Each stem was slightly wounded with a metal sponge and then sprayed with the bacterial suspension (108 CFU mL-1) of each isolate, and the experiment was repeated three times. Water-soaked symptoms were visible on the stems (Fig. 4E), while the control plants remained asymptomatic (Fig. 4F). The strains were then successfully re-isolated and identified by sequence analyses of their 16S ribosomal RNA gene and gyrB, icdA and proA genes. Therefore, the inoculation experiment of the isolatedbacterium fulfilled Koch's postulates. Previously, E. mori has been reported to cause bacterial wilt on white mulberry (Morus alba L.) (Zhu et al. 2022), peach fruit (Prunus persica) (Ahmad et al. 2021) and kiwifruit (Actinidia deliciosa [A. Chev.] CF Liang et AR Ferguson) (Zhang et al. 2021). To our knowledge, this is the first report of E. mori causing bacterial soft rot on watermelon in world.

PD31-04 LOWER URINARY TRACT MICROBIOTA DYSBIOSIS IS ASSOCIATED WITH URETHRAL FIBROSIS

You have accessJournal of UrologyCME1 May 2022PD31-04 LOWER URINARY TRACT MICROBIOTA DYSBIOSIS IS ASSOCIATED WITH URETHRAL FIBROSIS Michael Witthaus, Valmik Bhargava, Jill Buckley, and Mahadevan Rajasekaran Michael WitthausMichael Witthaus More articles by this author , Valmik BhargavaValmik Bhargava More articles by this author , Jill BuckleyJill Buckley More articles by this author , and Mahadevan RajasekaranMahadevan Rajasekaran More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002582.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cellular mechanisms of stricture progression after repeated transurethral interventions (TUI) are unclear. We hypothesize urethral injury and mechanical stretch can cause tears on the urethral epithelial cell lining leading to leaking epithelium and urine extravasation (Fig A). Urinary microbiota traverses the epithelial lining and fibrogenesis can be mediated by inflammation due to microbial metabolites. Our objective is to characterize the urinary microbiome of patients with urethral stricture disease (USD) using 16S rRNA taxonomic profiling to elucidate the clinical relevance of the urinary microbiome in USD progression. METHODS: First catch urine samples from USD patients and healthy volunteers were collected and frozen to −80°C. Prior to analysis, samples were thawed at 2-8°C and centrifuged at 4°C for 15 mins to form a pellet. The pellet was then used for bacterial genomic DNA extraction using the PowerMag Soil DNA Isolation Kit. PCR amplification of the V3 and V4 regions of the 16S rRNA gene was performed. Illumina sequencing adapters and unique dual-index barcodes were added by PCR. Samples were then pooled and sequenced with paired-end 150 base pair reads (PE150) on an Illumina MiSeq Instrument. Data was analyzed using Qiime, which was used to determine alpha and beta diversity metrics per sample and to determine which microbes were presented differentially between the two cohorts. RESULTS: Our results are summarized in Fig B-D. Visualizing the specific microbes using a relative abundance bar plot (Fig B), the microbiome of healthy urine was more diverse than patients with USD. A divergence between the two groups supported distinct microbiomes, and a significant difference between patients with USD and controls was observed with beta diversity analysis (Fig C, D). The findings were indicative of the overall composition of microbial communities in each patient population. CONCLUSIONS: Our findings suggest a potential role for urinary microbiota alterations in developing an inflammatory environment and consequent fibrosis progression in USD. Future studies will determine specific urinary microbes between the two cohorts and their relevance to USD. Further potential for targeting specific microbes using next-generation probiotics to correct the urinary microbiota dysbiosis may be beneficial in preventing stricture progression. Source of Funding: UCSD Academic Senate © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e542 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Michael Witthaus More articles by this author Valmik Bhargava More articles by this author Jill Buckley More articles by this author Mahadevan Rajasekaran More articles by this author Expand All Advertisement PDF DownloadLoading ...

R-Loop Detection in Bacteria.

Early evidence for R-loop formation in vivo came from the study of Escherichia coli topA (topoisomerase I; topo I) null mutants. Assays with plasmids to detect RNase HI-sensitive hypernegative supercoiling or R-looped DNA were used in vitro and in vivo to demonstrate R-loop formation. In addition, these R-loop-dependent topological modifications of plasmid DNA were shown to correlate with severe growth and gene expression inhibition in topA null mutants that could be corrected by RNase HI overproduction. However, direct evidence for R-loop formation on chromosomal DNA from E. coli cells was only obtained recently by using the S9.6 antibody to detect RNA-DNA hybrids in dot-blot experiments. Here, we present a protocol for such experiments with a special emphasis on the procedure used for bacterial genomic DNA extraction and preparation including treatment with appropriate ribonucleases to eliminate RNA-RNA hybrids (that are also recognized by S9.6) as well as single-stranded RNA (ssRNA), in order to obtain a signal that is specific to stable RNA-DNA hybrids generated. Furthermore, we recommend that the results of such experiments be correlated with RNase HI-sensitive phenotypes.

613 Vaginal carriage of haemophilus influenzae in a non-pregnant reproductive-age population

Haemophilus influenzae (Hi) is an emerging cause of early onset neonatal sepsis, but mechanisms of transmission are not well understood. We aimed to evaluate the prevalence of vaginal carriage of Hi in reproductive age women as a first step in assessing the role of peripartum vertical transmission in neonatal Hi disease. We performed a secondary analysis of stored vaginal lavage specimens from nonpregnant reproductive-age women. After extraction of bacterial genomic DNA, samples were tested for the presence of the gene encoding Haemophilus protein d (hpd) by quantitative real-time polymerase chain reaction (PCR) using validated primers and probe. PCR for the V3-V4 region of the 16S rRNA gene was performed as a positive control to assess sample quality. Samples with a cycle threshold (CT) value < 35 were defined as positive. Sanger sequencing confirmed the presence of hpd. We compared demographic and behavioral characteristics of women with and without vaginal carriage of Hi. Chi-square, Fisher’s exact, Mann-Whitney and t-test were used with p<0.05 set as the level of statistical significance. 415 samples were available for analysis. 315 (75.9%) had sufficient bacterial DNA present and were included in the analysis. Of these, 14 (4.4%) were positive for hpd. There were no demographic or behavioral differences between the women with Hi vaginal carriage and those without (Table 1). There was no difference in history of bacterial vaginosis, vaginal microbiome community state type, or presence of Group B Streptococcus (GBS) in women with and without vaginal carriage of Hi (Table 2). Haemophilus influenzae was present in vaginal lavage specimens of 4.4% of this cohort. Hi presence was unrelated to clinical or demographic characteristics, though the relatively small number of positive samples may have limited power to detect such differences. Further studies should assess for the presence of this possible perinatal pathogen in pregnant women and the rate of transmission to neonates.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Comparison of Swabbing Solution Volume and gDNA Extraction Kits on DNA Recovery from Rigid Surface.

For bacteria sampling studies, various collection methods have been used to identify bacteria. To obtain accurate information about bacteria, high quality samples should be obtained. In order to obtain a high quality sample, a stable and large number of DNA copies must be collected. This study compared the efficiency of different methods of bacterial gDNA extraction and bacteria collection according to swabbing solution volumes and types. The efficiency of bacterial genomic DNA extraction was compared using a AccuPrep® Genomic DNA Extraction kit, a QIAamp® DNA Mini kit, and a MOBIO® DNeasy PowerSoil kit. The DNA Mini kit was shown to extract the highest amount of gDNA, and sub-experiments were conducted using this kit. Phosphate-buffered saline and phosphate-buffered saline with 0.1% Tween 20 were used as collection solutions of various volumes (0, 40, 50, 60, 70, 80, 90, 100, 110, and 120μL) using cotton swabs. Bacteria collection efficiency was highest when 70μL PBS was used. The target strains collected in this experiment were Staphylococcus aureus and Escherichia coli, and these were quantified using the number of colony-forming units, DNA concentrations, and the number of DNA copies. These results can be used to efficiently bacterial collection for experiments in various fields.

Validation of a universal DNA extraction method for human and microbiAL DNA analysis

The study of microbiomes has enormous potential for forensic science because microorganisms are ubiquitous and particular communities of microbes are often associated with specific processes or environments. With recent advances in microbiome science, new opportunities exist for microbiome technologies in forensic science (PMI estimation, location of clandestine graves, soil analysis and personal identification). Before a new technology is accepted by the forensic science, it requires an initial validation phase.The aim of our study was to evaluate if the DNA IQ™ Casework Pro Kit for Maxwell® 16 (Promega) is suitable for microbial DNA extraction, without modifications. Ten bacterial strains were selected and subjected to the GenElute Bacterial Genomic DNA extraction protocol (Sigma-Aldrich) and to the DNA IQ™ Casework Pro Kit for Maxwell® 16 protocol. Extracted DNA was quantified and submitted to NGS analysis on an Ion S5 NGS System. Data were analyzed using the Ion Reporter Software metagenomics workflow. Our work has shown that it is possible to purify both microbial and human DNA using the Promega kit, thus making it possible to analyze both human and microbial DNA from a single trace, a pivotal factor in forensics where the quantities of biological material available are usually very limited.

Potential of Leucas zeylanica extract to eliminate E. coli and S. aureus in Corbicula fluminea (“Etak”) tissue

“Etak” or Corbicula fluminea, is a freshwater mollusc species regularly consumed as a popular snack among the Kelantanese in Malaysia. The “etak” is usually heated with traditional smoking process which is considered as half cooked and the smoked C. fluminea is commonly known as “etak salai”. This study focuses on the potential of Leucas zeylanica leaves extract to eliminate the bacteria content in “etak salai”. Extraction of bacterial genomic DNA was performed and confirmed the existence of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in “etak salai”. Antibacterial properties of L. zeylanica leaves extract was identified using disc diffusion assay and the result obtained exhibit that 70 μg/μL of L. zeylanica extract was the optimum concentration to give the effect of 11 mm inhibition zone for E. coli and 15 mm inhibition zone for S. aureus. This finding proof that L. zeylanica leaves could be the ingredients in the paste for “etak salai” preparation.

Ultra-High Efficient Colony PCR for High Throughput Screening of Bacterial Genes.

Current colony PCR methods are not suitable for screening genes encoded in genomic DNA and are limited to E. coli host strains. Here, we describe an ultra-high efficient colony PCR method for high throughput screening of bacterial genes embedded in the genomic DNA of any bacterial species. This new technique expands colony PCR method to several hosts as well as offers a rapid, less expensive and reliable bacterial genomic DNA extraction.

Gold Nanorod-based Photo-PCR System for One-Step, Rapid Detection of Bacteria.

The polymerase chain reaction (PCR) has been an essential tool for diagnosis of infectious diseases, but conventional PCR still has some limitations with respect to applications to point-of-care (POC) diagnostic systems that require rapid detection and miniaturization. Here we report a light-based PCR method, termed as photo-PCR, which enables rapid detection of bacteria in a single step. In the photo-PCR system, poly(enthylene glycol)-modified gold nanorods (PEG-GNRs), used as a heat generator, are added into the PCR mixture, which is subsequently periodically irradiated with a 808-nm laser to create thermal cycling. Photo-PCR was able to significantly reduce overall thermal cycling time by integrating bacterial cell lysis and DNA amplification into a single step. Furthermore, when combined with KAPA2G fast polymerase and cooling system, the entire process of bacterial genomic DNA extraction and amplification was further shortened, highlighting the potential of photo-PCR for use in a portable, POC diagnostic system.

Gut Microbiota and Metabolic Phenotype of Portunus Trituberculatus

16S rRNA gene high-throughput sequencing and nuclear magnetic resonance (NMR)-based metabolomics techniques were established for the composition analysis of gut microbiota and metabolites of Portunus trituberculatus. Swimming crab gut samples were used for the extraction of bacterial genomic DNA, followed by amplification of hypervariable domain V3-V4 of 16 S rRNA gene. The obtained PCR products were analyzed on an Illumina MiSeq platform. The bacterial phonotypes were then clustered and assigned. The results showed that swimming crab gut sample was dominated by Proteobacteria, Bacteroidetes, Fusobacteria, Tenericutes, and Acidobacteria at the phylum level, among which, Photobacterium, Paludibacter, and Propionigenium had the maximum abundance at the genus level. Crab intestinal samples were extracted with methanol/H2O (2:1, V/V) solution under shaking with a TissueLyser. After the removal of methanol, the resultant supernatants were lyophilized. Each of lyophilized extract was dissolved into Na+/K+ phosphate buffer and then centrifuged at high speed for NMR analysis. A standard Noesypr1D was used to acquire 1H NMR spectra. The 90° pulse length was adjusted to approximately 10 μs. The recycle delay and mixing time were set to 2 s and 100 ms in sequence. The spectral width was set to 20 ppm. Sixty-four transients were collected into 32768 data points for each spectrum. A range of two-dimensional NMR spectra were acquired for the resonance assignment. The results showed that swimming crab gut metabolome comprised 30 metabolites including some amino acids, organic acids and amines. In summary, this study provided a method for the systematic analyses of the compositions of gut bacterial community and metabolites of swimming crab.

Bacterial Diversity and Community Structure of Supragingival Plaques in Adults with Dental Health or Caries Revealed by 16S Pyrosequencing.

Dental caries has a polymicrobial etiology within the complex oral microbial ecosystem. However, the overall diversity and structure of supragingival plaque microbiota in adult dental health and caries are not well understood. Here, 160 supragingival plaque samples from patients with dental health and different severities of dental caries were collected for bacterial genomic DNA extraction, pyrosequencing by amplification of the 16S rDNA V1–V3 hypervariable regions, and bioinformatic analysis. High-quality sequences (2,261,700) clustered into 10,365 operational taxonomic units (OTUs; 97% identity), representing 453 independent species belonging to 122 genera, 66 families, 34 orders, 21 classes, and 12 phyla. All groups shared 7522 OTUs, indicating the presence of a core plaque microbiome. α diversity analysis showed that the microbial diversity in healthy plaques exceeded that of dental caries, with the diversity decreasing gradually with the severity of caries. The dominant phyla of plaque microbiota included Bacteroidetes, Actinobacteria, Proteobacteria, Firmicutes, Fusobacteria, and TM7. The dominant genera included Capnocytophaga, Prevotella, Actinomyces, Corynebacterium, Neisseria, Streptococcus, Rothia, and Leptotrichia. β diversity analysis showed that the plaque microbial community structure was similar in all groups. Using LEfSe analysis, 25 differentially abundant taxa were identified as potential biomarkers. Key genera (27) that potentially contributed to the differential distributions of plaque microbiota between groups were identified by PLS-DA analysis. Finally, co-occurrence network analysis and function predictions were performed. Treatment strategies directed toward modulating microbial interactions and their functional output should be further developed.

Effects of different carbohydrates on the simulation of human intestinal bacterial flora with in vitro culture

To investigate the optimal growth condition of human fecal bacterial flora in vitro by comparing the effect of different carbohydrates as cultural media. Three fecal samples (1, 2, 3) were collected and inoculated into a single-stage chemostat system, in which starch medium (VI) and starch polysaccharide medium(XP) were used. Samples were collected for bacterial genomic DNA extraction and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis. Bacterial composition and short chain fatty acid (SCFA) were then analyzed. The single stage chemostat system reached steady after operating 8 days, when evaluated by the PCR-DGGE. Bacterial 16s rRNA high-throughput sequencing showed that the intestinal bacteria of these three volunteers was mainly composed of four bacterial phyla, namely, Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria. When the influence of bacterial abundance was considered, the similarity of bacterial composition between the original fecal samples to the harvested flora after culture was 0.847, 0.825, 0.968 in VI medium and 0.927, 0.926, 0.836 in XP medium, respectively. The similarity was decreased to 0.553, 0.580, 0.623 with VI medium and 0.617, 0.520, 0.574 with XP medium, when the number of bacterial species was considered. The variation of host individual also influenced the simulation. VI medium favored fecal sample 3, while XP medium more benefited sample 1 and 2. Bacteroides and Lachnospiraceae_incertae_sedis grew in both VI and XP medium. However, some species were only detected in VI medium and some were specifically found in the XP medium. The SCFA concentration in fermenters was 15-35 mmol/L, mainly propionate and butyrate. The chemostat system works for stimulating human gut bacterial flora in vitro. The bacterial composition is affected by different carbohydrate in the culture medium yet with close simulation higher than 80%.

&amp;lt;i&amp;gt;mec&amp;lt;/i&amp;gt;A Gene Profile of Methicillin-Resistant &amp;lt;i&amp;gt;Staphylococcus aureus&amp;lt;/i&amp;gt; Isolates from Clinical Sources in Port Harcourt, Nigeria

<i>Staphylococcus aureus</i> strains are responsible for a range of acute to chronic infections in humans and other animals. There is scanty information about the genetic background of <i>S. aureus</i> strains in Rivers State, Nigeria. The aim of this study was to determine the occurrence of MRSA among <i>S. aureus</i> isolates as well as detect the presence of <i>mec</i>A gene among methicillin-resistant <i>Staphylococcus aureus</i> isolates in Port Harcourt, Nigeria. Two hundred and five (205) non duplicate <i>Staphylococcus aureus</i> previously isolated from human sources were randomly collected from three health facilities- University of Port Harcourt Teaching Hospital, Braithwaite Memorial Specialist Hospital and De-Integrated Laboratories- all located in Port Harcourt, Nigeria, for this study from August, 2012 to July, 2013. Isolates were grouped as hospital in-patient (Hospital-acquired – Nosocomial; n = 76) and out-patient cases (community-acquired; n = 129). Isolates were reconfirmed following standard laboratory protocols and stored in duplicate - one set at +4°C (for phenotypic detection of MRSA) and another set at -70°C for molecular analysis. Using the disk diffusion method, detection of MRSA was carried out with 1μg of oxacillin (OXOID) placed on Mueller-Hinton agar with 4% NaCl supplementation). <i>Molecular Analyses</i> were carried out on all ORSA strains as follows- Bacterial genomic DNA extraction and PCR amplification for detection of 16S rRNA and <i>mec</i>A genes. Amplified products were analyzed using 2.0% agarose gel electrophoresis and subsequently visualized on a UV trans-illuminator. About twelve percent (12.2%) of the 205 <i>Staphylococcus aureus</i> studied were resistant to oxacillin. MRSA detection was significantly higher in in-patient isolates (23.7% of 76) than out-patient (5.4% of 129) <i>S. aureus</i> (p = 0.00031). Urine samples accounted for majority of the isolates (52 of 205) but MRSA detection was highest in Wound swabs (9 of 48 isolates. Of the 25 MRSA, <i>mec</i>A gene was detected in 17, being significantly higher in in-patient MRSA (14) than out-patient MRSA (3) (p<0-05). This study has established the presence of the methicillin resistance encoding gene- <i>mec</i>A, among MRSA isolates in Port Harcourt and that this gene is largely responsible for the MRSA phenotype. Study further establishes that these MRSA are more frequent in the Hospital environment. Further studies on molecular epidemiology of <i>S. aureus</i> are recommended in this region. Improved infection control measures in the healthcare facilities as well as sustained surveillance of methicillin-resistant <i>S. aureus</i> in this region are also advocated.

Identification and isolation of bacteria containing OPH enzyme for biodegradation of organophosphorus pesticide diazinon from contaminated agricultural soil

Background: Organophosphorus insecticide diazinon has been widely used in agriculture and has the ability to transfer and accumulate in soil, water and animal tissues, and to induce toxicity in plants, animals and humans. In humans, diazinon inhibits nerve transmission by inactivating acetylcholinesterase enzyme. The present study was carried out to identify bacteria containing OPH enzyme for biodegradation of diazinon from contaminated agricultural soil. Methods: In this study, 8 contaminated agricultural soil samples that were exposed to pesticides, especially diazinon in the last two decades, were collected from the farms of Hamedan province. After preparing the media, for isolation of several bacterial strains containing OPH enzyme that are capable of biodegrading organophosphorus pesticides by diazinon enzymatic hydrolysis, bacterial genomic DNA extraction, plasmid product sequencing, phylogenetic sequence processing and phylogenetic tree drawing were carried out. Results: Eight bacterial strains, capable of secreting OPH enzyme, were isolated from soil samples, one of which named BS-1 with 86% similarity to Bacillus safensis displayed the highest organophosphate-hydrolyzing capability and can be used as a source of carbon and phosphorus. Conclusion: It can be concluded that the isolated bacterial strain identified in this study with OPH enzyme secretion has the potential for biodegradation of organophosphorus pesticides, especially diazinon in invitro conditions. Also, further studies such as the environmental stability and interaction, production strategies, safety, cost-benefit, environmental destructive parameters, and, toxicological, genetic and biochemical aspects are recommended prior to the application of bacterial strains in the field-scale bioremediation.