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Background: The lack of information regarding the burden of acute bacterial meningitis in Latin America leads to a reduction in the estimated incidence rates of the disease, and impairs public health decisions on the use and follow-up of preventive interventions, particularly, the evaluation of existing vaccination policies. The use of the real-time PCR in diagnostic routine procedures has resulted in a substantial increase in confirmed bacterial meningitis cases. However, in resource-poor countries, these assays are only available in reference laboratories. Sample transportation to these laboratories is a critical constraint, as it requires specialized, high cost courier services. To overcome this barrier we evaluated the use of FTATM Elute filter paper cards for the conservation and processing of samples under normal environmental conditions, as they would be when transported from remote and under-equipped healthcare facilities to the reference centers. A total of 401 samples received in 2015 as part of Sao Paulo’s national surveillance for routine diagnosis were selected for this study. Methods: The sensitivity and specificity of real-time PCR were evaluated using fresh serum and cerebrospinal fluid (CSF) samples processed using our laboratory’s standard DNA extraction, and processing the same samples after being dried and stored on FTATM card, and DNA extracted following the manufacturer’s instructions. Results: The sensitivities for detection of Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae from CSF dried and stored on FTATM cards were 98%, 92%, and 100%, respectively, and with serum samples were 73%, 88%, and 100%, respectively. When compared to our laboratory’s standard methodology, results showed high concordance, with Kappa index ranges of 0.9877–1.00 for CSF, and 0.8004–1.00 for serum samples. Conclusion: The use of FTATM cards for CSF and serum conservation and transport represents a rapid, reliable, and cost-effective alternative that will allow obtaining valuable epidemiological information that would otherwise be lost. [ABSTRACT FROM AUTHOR]

Pyocyanin is a toxin produced by Pseudomonas aeruginosa. Here we describe a novel paper-based electrochemical sensor for pyocyanin detection, manufactured with a simple and inexpensive approach based on electrode printing on paper. The resulting sensors constitute an effective electrochemical method to quantify pyocyanin in bacterial cultures without the conventional time consuming pretreatment of the samples. The electrochemical properties of the paper-based sensors were evaluated by ferri/ferrocyanide as a redox mediator, and showed reliable sensing performance. The paper-based sensors readily allow for the determination of pyocyanin in bacterial cultures with high reproducibility, achieving a limit of detection of 95 nM and a sensitivity of 4.30 μA/μM in standard culture media. Compared to the similar commercial ceramic based sensors, it is a 2.3-fold enhanced performance. The simple in-house fabrication of sensors for pyocyanin quantification allows researchers to understand in vitro adaptation of P. aeruginosa infections via rapid screenings of bacterial cultures that otherwise are expensive and time-consuming. [ABSTRACT FROM AUTHOR]

A system capable of biocatalytic conversion of distributed sources of single carbon gases such as carbon monoxide into hydrocarbons can be highly beneficial for developing commercially viable biotechnology applications in alternative energy. Several anaerobic bacterial strains can be used for such conversion. The anaerobic carbon monoxide-fixing bacteria Clostridium ljungdahlii OTA1 is a model CO assimilating microorganism that currently requires cryogenic temperature for storage of the viable strains. If these organisms can be stabilized and concentrated in thin films in advanced porous materials, it will enable development of high gas fraction, biocomposite absorbers with elevated carbon monoxide (CO) mass transfer rate, that require minimal power input and liquid, and demonstrate elevated substrate consumption rate compared to conventional suspended cell bioreactors. We report development of a technique for dry-stabilization of C. ljungdahlii OTA1 on a paper biocomposite. Bacterial samples coated onto paper were desiccated in the presence of trehalose using convective drying and stored at 4°C. Optimal dryness was ~1g H2O per gram of dry weight (gDW). CO uptake directly following biocomposite rehydration steadily increases over time indicating immediate cellular metabolic recovery. A high-resolution Raman microspectroscopic hyperspectral imaging technique was employed to spatially quantify the residual moisture content. We have demonstrated for the first time that convectively dried and stored C. ljungdahlii strains were stabilized in a desiccated state for over 38 days without a loss in CO absorbing reactivity. The Raman hyperspectral imaging technique described here is a non-invasive characterization tool to support development of dry-stabilization techniques for microorganisms on inexpensive porous support materials. The present study successfully extends and implements the principles of dry-stabilization for preservation of strictly anaerobic bacteria as an alternative to lyophilization or spray drying that could enable centralized biocomposite biocatalyst fabrication and decentralized bioprocessing of CO to liquid fuels or chemicals. [ABSTRACT FROM AUTHOR]

H-index is the most commonly applied tool to evaluate scientific productivity. In this study, the use of the H-index to evaluate scientific production in swine veterinary medicine was explored. A database of 137 pig infectious agents was constructed, including its taxonomic division, zoonotic potential, status as emerging pathogen and whether it was OIE-listed. The H-index and the total number of citations were calculated for those pathogens, the location of the affiliation of the first author of each paper included in the H-index core was registered and, for the ten pathogens with the highest H-index, evolution over time was measured. H-index values were compared to the M quotient, A-index, G-index, HG-index and the G/H ratio. H-indices were found to be severely affected by search accuracy and the database was hand curated. Swine pathogen H-indexes were highly dispersed ranging from 0 to 106 and were generally higher for pathogens causing endemic diseases in large pig producing countries. Indeed, the three top pathogens were Escherichia coli, Porcine reproductive and respiratory syndrome virus and Porcine circovirus type 2 with H-indices 106, 95 and 85, respectively. H-indices of viruses and bacteria were significantly higher (P<0.001) than other pathogen types. Also, non-zoonotic pathogens had higher H-indices than zoonotic pathogens (p<0.009) while no differences could be found for being listed by the OIE. For emerging diseases, only non-emerging viruses had higher H-index (p = 0.02). The study of H-indexes over time revealed three general patterns and that they had increased mainly after the 1980’s. As expected, there were strong geographic patterns in terms of authorship and North America (38%) and Europe (46%) coped the majority of the papers. Finally, in order to quantify the contribution of a subject to a specific field, a new index “Deciphering Citations Organized by Subject” (Dcos) is proposed. [ABSTRACT FROM AUTHOR]

Cholera rapid diagnostic tests (RDT) could play a central role in outbreak detection and surveillance in low-resource settings, but their modest performance has hindered their broad adoption. The addition of an enrichment step may improve test specificity. We describe the results of a prospective diagnostic evaluation of the Crystal VC RDT (Span Diagnostics, India) with enrichment step and of culture, each compared to polymerase chain reaction (PCR), during a cholera outbreak in South Sudan. RDTs were performed on alkaline peptone water inoculated with stool and incubated for 4–6 hours at ambient temperature. Cholera culture was performed from wet filter paper inoculated with stool. Molecular detection of Vibrio cholerae O1 by PCR was done from dry Whatman 903 filter papers inoculated with stool, and from wet filter paper supernatant. In August and September 2015, 101 consecutive suspected cholera cases were enrolled, of which 36 were confirmed by PCR. The enriched RDT had 86.1% (95% CI: 70.5–95.3) sensitivity and 100% (95% CI: 94.4–100) specificity compared to PCR as the reference standard. The sensitivity of culture versus PCR was 83.3% (95% CI: 67.2–93.6) for culture performed on site and 72.2% (95% CI: 54.8–85.8) at the international reference laboratory, where samples were tested after an average delay of two months after sample collection, and specificity was 98.5% (95% CI: 91.7–100) and 100% (95% CI: 94.5–100), respectively. The RDT with enrichment showed performance comparable to that of culture and could be a sustainable alternative to culture confirmation where laboratory capacity is limited. [ABSTRACT FROM AUTHOR]

Background: Nigeria has one of the highest burdens of pneumococcal disease in the world, but accurate surveillance is lacking. Molecular detection of infectious pathogens in dried blood spots (DBS) is an ideal method for surveillance of infections in resource-limited settings because of its low cost, minimal blood volumes involved, and ease of storage at ambient temperature. Our study aim was to evaluate a Streptococcus pneumoniae real-time polymerase chain reaction (rt-PCR) assay on DBS from febrile Nigerian children on Whatman 903 and FTA filter papers, compared to the gold standard of culture. Methods: Between September 2011 to May 2015, blood was collected from children 5 years of age or under who presented to six hospital study sites throughout northern and central Nigeria with febrile illness, and inoculated into blood culture bottles or spotted onto Whatman 903 or FTA filter paper. Culture and rt-PCR were performed on all samples. Results: A total of 537 DBS specimens from 535 children were included in the study, of which 15 were culture-positive for S. pneumoniae. The rt-PCR assay detected S. pneumoniae in 12 DBS specimens (2.2%). One positive rt-PCR result was identified in a culture-negative specimen from a high-risk subject, and two positive rt-PCR results were negative on repeat testing. Six culture-confirmed cases of S. pneumoniae bacteremia were missed. Compared to culture, the overall sensitivities of Whatman 903 and FTA DBS for detection of S. pneumoniae were 57.1% (95% CI 18.4–90.1%) and 62.5% (95% CI 24.5–91.5%), respectively. Nonspecific amplification was noted in an additional 22 DBS (4.1%). Among these, six were positive for a non-S. pneumoniae pathogen on culture. Conclusions: Rt-PCR was able to detect S. pneumoniae from clinical DBS specimens, including from a culture-negative specimen. Our findings show promise of this approach as a surveillance diagnostic, but also raise important cautionary questions. Several DBS specimens were detected as S. pneumoniae by rt-PCR despite growth of a non-S. pneumoniae pathogen on culture. A precise definition of what constitutes a positive result is required to avoid falsely over-identifying specimens. [ABSTRACT FROM AUTHOR]

Introduction: Despite evidence of the high prevalence of antibiotic resistant infections in developing countries, studies on the clinical and economic impact of antibiotic resistance (ABR) to inform interventions to contain its emergence and spread are limited. The aim of this study was to analyze the published literature on the clinical and economic implications of ABR in developing countries. Methods: A systematic search was carried out in Medline via PubMed and Web of Sciences and included studies published from January 01, 2000 to December 09, 2016. All papers were considered and a quality assessment was performed using the Newcastle-Ottawa quality assessment scale (NOS). Results: Of 27 033 papers identified, 40 studies met the strict inclusion and exclusion criteria and were finally included in the qualitative and quantitative analysis. Mortality was associated with resistant bacteria, and statistical significance was evident with an odds ratio (OR) 2.828 (95%CI, 2.231–3.584; p = 0.000). ESKAPE pathogens was associated with the highest risk of mortality and with high statistical significance (OR 3.217; 95%CIs; 2.395–4.321; p = 0.001). Eight studies showed that ABR, and especially antibiotic-resistant ESKAPE bacteria significantly increased health care costs. Conclusion: ABR is associated with a high mortality risk and increased economic costs with ESKAPE pathogens implicated as the main cause of increased mortality. Patients with non-communicable disease co-morbidities were identified as high-risk populations. [ABSTRACT FROM AUTHOR]

SCCmec elements are very important mobile genetic elements in Staphylococci that carry beta-lactam resistance genes mecA/mecC, recombinase genes and a variety of accessory genes. Twelve main types and a couple of variants have yet been described. In addition, there are also other SCC elements harbouring other markers. In order to subtype strains of methicillin-resistant S. aureus (MRSA) based on variations within their SCCmec elements, 86 markers were selected from published SCC sequences for an assay based on multiplexed primer extension reactions followed by hybridisation to the specific probes. These included mecA/mecC, fusC, regulatory genes, recombinase genes, genes from ACME and heavy metal resistance loci as well as several genes of unknown function. Hybridisation patterns for published genome or SCC sequences were theoretically predicted. For validation of the microarray based assay and for stringent hybridisation protocol optimization, real hybridization experiments with fully sequenced reference strains were performed modifying protocols until yielded the results were in concordance to the theoretical predictions. Subsequently, 226 clinical isolates from two hospitals in the city of Dresden, Germany, were characterised in detail. Beside previously described types and subtypes, a wide variety of additional SCC types or subtypes and pseudoSCC elements were observed as well as numerous composite elements. Within the study collection, 61 different such elements have been identified. Since hybridisation cannot recognise the localisation of target genes, gene duplications or inversions, this is a rather conservative estimate. Interestingly, some widespread epidemic strains engulf distinct variants with different SCCmec subtypes. Notable examples are ST239-MRSA-III, CC5-, CC22-, CC30-, and CC45-MRSA-IV or CC398-MRSA-V. Conversely, identical SCC elements were observed in different strains with SCCmec IVa being spread among the highest number of Clonal Complexes. The proposed microarray can help to distinguish isolates that appear similar or identical by other typing methods and it can be used as high-throughput screening tool for the detection of putative new SCC types or variants that warrant further investigation and sequencing. The high degree of diversity of SCC elements even within so-called strains could be helpful for epidemiological typing. It also raises the question on scale and speed of the evolution of SCC elements. [ABSTRACT FROM AUTHOR]

Purpose: This systematic review was conducted to gain insight into the efficacy of transmission of infectious agents to colony sentinels by soiled bedding transfer based on publications studying this subject in mice and rats. This information is essential to establish recommendations for the design of health monitoring programs which use sentinels to determine the microbiological status of laboratory animal colonies. Results: Fifteen original articles retrieved from PubMed, Embase, and CAB abstracts met the inclusion criteria. The design of the studies varied substantially per infectious agent with regard to dose of soiled bedding, exposure time, and sentinel strains used. Conclusion: With our conservative criteria for effectiveness, soiled bedding transfer appeared to be effective for MHV, MPV, TMEV, Helicobacter spp., and fur mite infections and ineffective for Sendai virus. For other infectious agents, such as MNV, EDIM, MVM, SDAV, Clostridium piliforme, and pinworms, too few data were available to be able to draw robust conclusions on the efficacy of soiled bedding transfer. Recommendation: The identified evidence only pertains to a portion of the infectious organisms included in the FELASA 2014 guidelines. As many animal facilities design their health monitoring program according to these recommendations, additional studies are warranted to draw comprehensive conclusions on the effective transmission of the infectious agents listed in these guidelines by soiled bedding transfer. [ABSTRACT FROM AUTHOR]

A photo-medical capsule that emits blue light for Helicobacter pylori treatment was described in this paper. The system consists of modules for pH sensing and measuring, light-emitting diode driver circuit, radio communication and microcontroller, and power management. The system can differentiate locations by monitoring the pH values of the gastrointestinal tract, and turn on and off the blue light according to the preset range of pH values. Our experimental tests show that the capsule can operate in the effective light therapy mode for more than 32 minutes and the wireless communication module can reliably transmit the measured pH value to a receiver located outside the body. [ABSTRACT FROM AUTHOR]

Nucleases are enzymes that can degrade genomic DNA and RNA that decrease the accuracy of quantitative measures of those nucleic acids. Here, we study conventional heating, standard microwave irradiation, and Lyse-It, a microwave-based lysing technology, for the potential to fragment and inactivate DNA and RNA endonucleases. Lyse-It employs the use of highly focused microwave irradiation to the sample ultimately fragmenting and inactivating RNase A, RNase B, and DNase I. Nuclease size and fragmentation were determined visually and quantitatively by SDS polyacrylamide gel electrophoresis and the mini-gel Agilent 2100 Bioanalyzer system, with a weighted size calculated to depict the wide range of nuclease fragmentation. Enzyme activity assays were conducted, and the rates were calculated to determine the effect of various lysing conditions on each of the nucleases. The results shown in this paper clearly demonstrate that Lyse-It is a rapid and highly efficient way to degrade and inactivate nucleases so that nucleic acids can be retained for down-stream detection. [ABSTRACT FROM AUTHOR]

The survey described in this research paper aimed to investigate the economic and health impacts of birds on dairies. Birds are common pests on dairies, consuming and contaminating feed intended for cattle. As a result, dairy operators experience increased feed costs and increased pathogen and disease risk. We surveyed dairy operators attending the 2017 Washington Dairy Conference to examine the impact of birds on dairies in Washington State. Dairy operators reported feed losses valued at $55 per cow resulting in annual losses totaling $5.5 million in the Western region of the state and $9.2 million in the Eastern region of the state. Shooting was the most commonly used bird management method and European starlings (Sternus vulgaris) were the most frequently implicated species statewide. Bird abundance greater than 10,000 birds per day was associated with larger herd size and with self-reported presence of Johne’s disease and Salmonella. [ABSTRACT FROM AUTHOR]

Sigma factors are key proteins that mediate the recruitment of RNA polymerase to the promoter regions of genes, for the initiation of bacterial transcription. Multiple sigma factors in a bacterium selectively recognize their cognate promoter sequences, thereby inducing the expression of their own regulons. In this paper, we report the crystal structure of the σ4 domain of Bacillus subtilis SigW bound to the -35 promoter element. Purine-specific hydrogen bonds of the -35 promoter element with the recognition helix α9 of the σ4 domain occurs at three nucleotides of the consensus sequence (G-35, A-34, and G’-31 in G-35A-34A-33A-32C-31C-30T-29). The hydrogen bonds of the backbone with the α7 and α8 of the σ4 domain occurs at G’-30. These results elucidate the structural basis of the selective recognition of the promoter by SigW. In addition, comparison of SigW structures complexed with the -35 promoter element or with anti-sigma RsiW reveals that DNA recognition and anti-sigma factor binding of SigW are mutually exclusive. [ABSTRACT FROM AUTHOR]

Image segmentation applied to medical image analysis is still a critical and important task. Although there exist several segmentation algorithms that have been widely studied in literature, these are subject to segmentation problems such as over- and under-segmentation as well as non-closed edges. In this paper, a simple method that combines well-known segmentation algorithms is presented. This method is applied to detect acid-fast bacilli (AFB) in bacilloscopies used to diagnose pulmonary tuberculosis (TB). This diagnosis can be performed through different tests, and the most used worldwide is smear microscopy because of its low cost and effectiveness. This diagnosis technique is based on the analysis and counting of the bacilli in the bacilloscopy observed under an optical microscope. The proposed method is used to segment the bacilli in digital images from bacilloscopies processed using Ziehl-Neelsen (ZN) staining. The proposed method is fast, has a low computational cost and good efficiency compared to other methods. The bacilli image segmentation is performed by image processing and analysis techniques, probability concepts and classifiers. In this work, a Bayesian classifier based on a Gaussian mixture model (GMM) is used. The segmentations' results are validated by using the Jaccard index, which indicates the efficiency of the classifier. [ABSTRACT FROM AUTHOR]

Streptococcus pneumonia is an important human pathogen that causes various severe diseases such as pneumonia, otitis and meningitis. Vaccination against S. pneumoniae is implemented in many developed countries. The presently used vaccines are safe, well tolerated but relatively expensive and require modification due to the immunological changes of the epidemic strains. This paper describes the development of a new pneumococcal vaccine candidate for immunization on mucosal surfaces. For this purpose the antigens of chimeric protein PSPF, previously suggested for an injectable S. pneumoniae vaccine, were expressed on the surface of the live probiotic strain Enterococcus faecium L3. Experiments on laboratory mice vaccinated with live bacteria demonstrated the appearance of the specific IgA and IgG which provide protection against the lethal S. pneumoniae infection. [ABSTRACT FROM AUTHOR]

This paper reports an outdoor-use polymerase chain reaction (PCR) technology in which stability of resistance temperature detectors (RTDs) is remarkably improved. A thin-film RTD made of non-annealed Pt shows accuracy degradation because the resistance of the RTD tends to decrease during the PCR operation. Thus, the annealing process is applied to the Pt RTD to improve the stability, which is a prerequisite to the accurate measurement of the absolute temperature. Both heaters and the RTD are fabricated on a thin quartz substrate whose melting temperature is high enough for annealing. The performances in the PCR time and power consumption are enhanced by reducing the size of the heater chips with no degradation in the temperature uniformity. A spring-loaded electrode is employed to simplify the procedure of electrical connection to the thermal controller and loading/unloading of the PCR chip. The contact area of the electrical connection is so small that the conductive thermal resistance increases; thereby small heat dissipation can be exploited for low-power operation. The stability of the RTD is experimentally confirmed in terms of resistance variation over repeated PCR operations (four times). The least variation of 0.005%, which corresponds to a negligible temperature variation of 0.038 °C for the PCR, is achieved from the RTD annealed for 5 min at 450 °C. The gel-electrophoresis result indicates that the PCR performance of the proposed system using a film-type PCR chip is comparable to that of a conventional system using a vial tube despite its low power consumption. [ABSTRACT FROM AUTHOR]

Biofilm-related infections of bones pose a significant therapeutic issue. In this article we present in vitro results of the efficacy of gentamicin released from a collagen sponge carrier against Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae biofilms preformed on hydroxyapatite surface. The results indicate that high local concentrations of gentamicin released from a sponge eradicate the biofilm formed not only by gentamicin-sensitive strains but, to some extent, also by those that display a resistance pattern in routine diagnostics. The data presented in this paper is of high clinical translational value and may find application in the treatment of bone infections. [ABSTRACT FROM AUTHOR]

Leptospirosis is probably the most widespread zoonotic disease in the world especially in tropical countries. There has been an increase in individual studies, which assessed the frequency of leptospirosis in flood conditions. Some studies showed contact with floods was significantly associated with the occurrence of leptospirosis while other studies reported differently. The objective of this meta-analysis was to synthesize the evidence on the risk factors which are associated with human leptospirosis following flooding. We set up the inclusion criteria and searched for the original studies, addressing leptospirosis in human with related to flood in health-related electronic databases including PubMed, Embase, Ovid Medline, google scholar and Scopus sources. We used the terms ‘leptospirosis’, ‘flood’, ‘risk factor’ and terms from the categories were connected with “OR” within each category and by “AND” between categories. The initial search yielded 557 citations. After the title and abstract screening, 49 full-text papers were reviewed and a final of 18 observational studies met the pre-specified inclusion criteria. Overall, the pooled estimates of 14 studies showed that the contact with flooding was a significant factor for the occurrence of leptospirosis (pooled OR: 2.19, 95%CI: 1.48–3.24, I2:86%). On stratification, the strength of association was greater in the case-control studies (pooled OR: 4.01, 95%CI: 1.26–12.72, I2:82%) than other designs (pooled OR:1.77,95%CI:1.18–2.65, I2:87%). Three factors such as ‘being male’(pooled OR:2.06, 95%CI:1.29–2.83), the exposure to livestock animals (pooled OR: 1.95, 95%CI:1.26–2.64), the lacerated wound (pooled OR:4.35, 95%CI:3.07–5.64) were the risk factors significantly associated with the incidence of leptospirosis following flooding in the absence of within-study heterogeneity (I2: 0%). We acknowledge study limitations such as publication bias and type 2 statistical errors. We recommended flood control and other environmental modifications that are expected to reduce the risk of leptospiral infection, and a multi-sectoral effort to this aspect would have long-term benefits. [ABSTRACT FROM AUTHOR]

Aims: Enhanced Biological Phosphorus Removal (EBPR) is a technology widely used in wastewater treatment to remove phosphorus (P) and prevent eutrophication. Establishing its operating efficiency and stability is an active research field that has generated almost 3000 publications in the last 40 years. Due to its size, including over 119 review articles, it is an example of a field where it becomes increasingly difficult to manually recognize its key research contributions, especially for non-experts or newcomers. Therefore, this work included two distinct but complementary objectives. First, to assemble for the first time a collection of bibliometric techniques into a framework for automating the article selection process when preparing a literature review (section 2). Second, to demonstrate it by applying it to the field of EBPR, producing a bibliometric analysis and a review of the key findings of EBPR research over time (section 3). Findings: The joint analysis of citation networks, keywords, citation profiles, as well as of specific benchmarks for the identification of highly-cited publications revealed 12 research topics. Their content and evolution could be manually reviewed using a selection of articles consisting of approximately only 5% of the original set of publications. The largest topics addressed the identification of relevant microorganisms, the characterization of their metabolism, including denitrification and the competition between them (Clusters A-D). Emerging and influential topics, as determined by different citation indicators and temporal analysis, were related to volatile fatty acid production, P-recovery from waste activated sludge and aerobic granules for better process efficiency and stability (Clusters F-H). Conclusions: The framework enabled key contributions in each of the constituent topics to be highlighted in a way that may have otherwise been biased by conventional citation-based ranking. Further, it reduced the need for manual input and a priori expertise compared to a traditional literature review. Hence, in an era of accelerated production of information and publications, this work contributed to the way that we are able to use computer-aided approaches to curate information and manage knowledge. [ABSTRACT FROM AUTHOR]

Background and aims: The bacterial leaf nodule symbiosis is a close interaction between endophytes and their plant hosts, mainly within the coffee family. The interaction between Rubiaceae species and Burkholderia bacteria is unique due to its obligate nature, high specificity, and predominantly vertical transmission of the endophytes to the next generation of host plants. This vertical transmission is intriguing since it is the basis for the uniqueness of the symbiosis. However, unequivocal evidence of the location of the endophytes in the seeds is lacking. The aim of this paper is therefore to demonstrate the presence of the host specific endophyte in the seeds of Psychotria punctata and confirm its precise location. In addition, the suggested location of the endophyte in other parts of the host plant is investigated. Methods: To identify and locate the endophyte in Psychotria punctata, a two-level approach was adopted using both a molecular screening method and fluorescent in situ hybridisation microscopy. Key results: The endophytes, molecularly identified as Candidatus Burkholderia kirkii, were detected in the leaves, vegetative and flower buds, anthers, gynoecium, embryos, and young twigs. In addition, they were in situ localised in leaves, flowers and shoot apical meristems, and, for the first time, in between the cotyledons of the embryos. Conclusions: Both independent techniques detected the host specific endophyte in close proximity to the shoot apical meristem of the embryo, which confirms for the first time the exact location of the endophytes in the seeds. This study provides reliable proof that the endophytes are maintained throughout the growth and development of the host plant and are transmitted vertically to the offspring. [ABSTRACT FROM AUTHOR]

Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections. [ABSTRACT FROM AUTHOR]

Salmonella Typhi and Salmonella Paratyphi A are the leading causative agents of enteric fever which cause morbidity and mortality worldwide. Currently, there is no combination vaccine which could protect infection from both the strains. In this paper, we are focusing on the development of a novel bivalent typhoidal Outer Membrane Vesicles (OMVs) based immunogen against enteric fever. We have isolated Salmonella Typhi and Paratyphi A OMVs and also characterized OMVs associated antigens. Then we immunized adult mice with three doses of our newly formulated bivalent immunogen orally (25 μg/200 μl). After three doses of oral immunization, we found our immunogen could significantly induce humoral response. We have also found serum IgG against LPS, Vi-polysaccharide etc. OMV immunization induces CD4, CD8 and CD19 population in immunized mice spleen. It also induces Th1 and Th17-cell mediated immunity. We also found bivalent OMVs immunization can prevent more than lethal dose of heterologous Salmonella strains mediated systemic infection in adult mice model. We determined that, the protective immune responses depend on the humoral and cell-mediated immune response. Furthermore, we have evaluated the mode of protective immune response carried out by anti-OMVs antibody by significantly inhibiting bacterial motility and mucin penetration ability. Taken together, these findings suggest that our bivalent immunogen could be used as a novel candidate vaccine against enteric fever. [ABSTRACT FROM AUTHOR]

It is well accepted that influenza A virus predisposes individuals to often more severe superinfections with Streptococcus pneumonia. However, the mechanisms that lead to this synergy are not clearly understood. Recent data suggests that competent Th17 immunity is crucial to clearance and protection from invasive pneumococcal disease of the lung. We demonstrate that early influenza infection significantly reduced levels of pneumococcus driven IL-12p70, IL-23 and IL-27 in human monocytes with significant impairment of IL-17A and IFN-γ in HKSP-treated allogeneic mixed lymphocyte cultures. We also provide evidence to suggest that the hemagglutinin component of the virus is at least partially responsible for this downward pressure on IL-17 responses but surprisingly this suppression occurs despite robust IL-23 levels in hemagglutinin-treated monocyte cultures. This study demonstrates that influenza can directly affect the immunological pathways that promote appropriate responses to Streptococcus pneumonia in human immune cells. Importance: Influenza virus is highly contagious and poses substantial public health problems due to its strong association with morbidity and mortality. Approximately 250,000–500,000 deaths are caused by seasonal influenza virus annually, and this figure increases during periods of pandemic infections. Most of these deaths are due to secondary bacterial pneumonia. Influenza-bacterial superinfection can result in hospitalisation and/or death of both patients with pre-existing lung disease or previously healthy individuals. The importance of our research is in determining that influenza and its component haemagglutinin has a direct effect on the classic pneumococcus induced pathways to IL-17A in our human ex vivo model. Our understanding of the mechanism which leaves people exposed to influenza infection during superinfection remain unresolved. This paper demonstrates that early infection of monocytes inhibits an arm of immunity crucial to bacterial clearance. Understanding this mechanism may provide alternative interventions in the case of superinfection with antimicrobial resistant strains of bacteria. [ABSTRACT FROM AUTHOR]

Sample preparation is a leading bottleneck in rapid detection of pathogenic bacteria. Here, we use Lyse-It® for bacterial cellular lysis, genomic DNA fragmentation, and protein release and degradation for both Listeria monocytogenes and Vibrio cholerae. The concept of Lyse-It® employs a conventional microwave and Lyse-It® slides for intensely focused microwave irradiation onto the sample. High microwave power and a <60 second irradiation time allow for rapid cellular lysis and subsequent intracellular component release. The pathogenic bacteria are identified by quantitative polymerase chain reaction (qPCR), which subsequently demonstrates the viability of DNA for amplification post microwave-induced lysis. Intracellular component release, degradation, and detection of L. monocytogenes and V. cholerae has been performed and shown in this paper. These results demonstrate a rapid, low-cost, and efficient way for bacterial sample preparation on both food and water-borne Gram-positive and -negative organisms alike. [ABSTRACT FROM AUTHOR]

Endophytic strains were isolated from different parts of a healthy “Dongzao” jujube (Ziziphus jujuba Mill. ‘Dongzao’) to find biocontrol agents against jujube shrunken-fruit disease caused by Alternaria alternata. The strains were screened using A. alternata strain CN193 as the target pathogen. The nutrient competition for all isolates was studied using the dual culture, and their inhibitive capability was tested by measuring the inhibition width of filter paper disks with filtrate. Influence of filtrate from the selected strains with strong inhibition of mycelial growth on spore germination was studied with hanging drop method on concavity slides. Colonization in the jujube leaves was assayed using a rifampicin-resistant mutant of strain St-zn-34 as the screening marker. Strains were identified based on their morphological, physiological, and biochemical characteristics, 16S rDNA sequencing, and phylogenetic analysis. A total of 81 endophytic strains were isolated from the stems, leaves, flowers, and fruits of winter jujube. Among these isolates, 14 strains showed strong antagonism against A. alternata. Further study showed that the filtrate of strains St-zn-9 and St-zn-34 could inhibit the mycelial growth of A. alternata, and the widths of their inhibition zone reached 6.14±0.03 mm and 8.27±0.09 mm, respectively. However, strain St-zn-34 showed stronger inhibition on spore germination than strain St-zn-9. St-zn-34 could significantly reduce the spore germination rate of A. alternata, and the spore did not germinate at all or the germ tube was very short. A rifampicin resistant-derivative of wild-type strain St-zn-34, which was designated as St-zn-34r, was obtained by transferring the strains to media with stepwise-increased rifampicin. Colonization assays indicated that St-zn-34r could colonize in jujube leaves, and the population of St-zn-34r was 1.2×103 CFU/g FW after inoculation for 30 days. Except for its salt tolerance, St-zn-34 was the closest to those of Bacillus subtilis. Thus, the strain was identified as B. subtilis. [ABSTRACT FROM AUTHOR]

Epidemiological studies use saliva on a regular basis as a non-invasive and easy-to-take sample, which is assumed to be a microbial representative of the oral cavity ecosystem. However, comparative studies between different kinds of saliva samples normally used in microbial studies are scarce. The aim of the current study was to compare oral microbiota composition between two different saliva samples collected simultaneously: non-stimulated saliva with paper points and stimulated saliva collected after chewing paraffin gum. DNA was extracted from saliva samples of ten individuals, then analyzed by 16S rRNA pyrosequencing to describe bacterial diversity. The results demonstrate significant differences between the microbiota of these two kinds of saliva. Stimulated saliva was found to contain an estimated number of species over three times higher than unstimulated saliva. In addition, bacterial composition at the class and genus level was radically different between both types of samples. When compared to other oral niches, both types of saliva showed some similarity to tongue and buccal mucosa, but they do not correlate at all with the bacterial composition described in supra- or sub-gingival dental plaque, questioning their use in etiological and epidemiological studies of oral diseases of microbial origin. [ABSTRACT FROM AUTHOR]

Type IV secretion systems (T4SS) are multi-protein complexes in a number of bacterial pathogens that can translocate proteins and DNA to the host. Most T4SSs function in conjugation and translocate DNA; however, approximately 13% function to secrete proteins, delivering effector proteins into the cytosol of eukaryotic host cells. Upon entry, these effectors manipulate the host cell’s machinery for their own benefit, which can result in serious illness or death of the host. For this reason recognition of T4SS effectors has become an important subject. Much previous work has focused on verifying effectors experimentally, a costly endeavor in terms of money, time, and effort. Having good predictions for effectors will help to focus experimental validations and decrease testing costs. In recent years, several scoring and machine learning-based methods have been suggested for the purpose of predicting T4SS effector proteins. These methods have used different sets of features for prediction, and their predictions have been inconsistent. In this paper, an optimal set of features is presented for predicting T4SS effector proteins using a statistical approach. A thorough literature search was performed to find features that have been proposed. Feature values were calculated for datasets of known effectors and non-effectors for T4SS-containing pathogens for four genera with a sufficient number of known effectors, Legionella pneumophila, Coxiella burnetii, Brucella spp, and Bartonella spp. The features were ranked, and less important features were filtered out. Correlations between remaining features were removed, and dimensional reduction was accomplished using principal component analysis and factor analysis. Finally, the optimal features for each pathogen were chosen by building logistic regression models and evaluating each model. The results based on evaluation of our logistic regression models confirm the effectiveness of our four optimal sets of features, and based on these an optimal set of features is proposed for all T4SS effector proteins. [ABSTRACT FROM AUTHOR]

Introduction: Sexually Transmitted Infections (STIs) have always represented a public health concern in the military, yet most studies rely on self-reports among non-random samples of military populations. In addition, most of the studies exploring STI rates among the military focus on US service members. This paper assesses the prevalence and correlates of STIs in the French military using biomarkers and compares self-reported versus diagnosed STIs. Methods: Data are drawn from the COSEMIL study, a national sexual health survey conducted in the French military in 2014 and 2015. A random sample of 784 men and 141 women aged 18–57 years completed a self-administered questionnaire and provided biological samples for STI testing. We used logistic regression modeling to identify the correlates of STI diagnosis and self-reports. Results: The prevalence of diagnosed STIs was 4.7% [3.8–5.9], mostly due to Chlamydia trachomatis. This rate was four times higher than the 12 months self-reported rate of 1.1% [0.6–2.3]. Reported STI rates were similar among men and women (1.1% versus 1.8%), but diagnosed STI rates were twice as high among females versus males (10.4% versus 4.1%, p = 0.007). There were significant differences in the determinants of reported versus diagnosed STIs. In particular, age and sexual orientation were associated with reported STIs, but not with diagnosed STIs. Conversely, STI counseling and depression were associated with STI diagnosis but not with STI reports. Conclusion: This study underlines the need to use biomarkers in population-based surveys, given the differential and substantial underreporting of STIs. Results also highlight the need for programmatic adaptation to address gender inequalities in STI rates, by developing women’s health services in the French military. Addressing such needs not only benefits women but could also serve as a strategy to reduce overall STI rates as most military women have military partners, increasing the risk of internal transmission. [ABSTRACT FROM AUTHOR]

Norovirus is a major cause of viral gastroenteritis, with shellfish consumption being identified as one potential norovirus entry point into the human population. Minimising shellfish norovirus levels is therefore important for both the consumer’s protection and the shellfish industry’s reputation. One method used to reduce microbiological risks in shellfish is depuration; however, this process also presents additional costs to industry. Providing a mechanism to estimate norovirus levels during depuration would therefore be useful to stakeholders. This paper presents a mathematical model of the depuration process and its impact on norovirus levels found in shellfish. Two fundamental stages of norovirus depuration are considered: (i) the initial distribution of norovirus loads within a shellfish population and (ii) the way in which the initial norovirus loads evolve during depuration. Realistic assumptions are made about the dynamics of norovirus during depuration, and mathematical descriptions of both stages are derived and combined into a single model. Parameters to describe the depuration effect and norovirus load values are derived from existing norovirus data obtained from U.K. harvest sites. However, obtaining population estimates of norovirus variability is time-consuming and expensive; this model addresses the issue by assuming a ‘worst case scenario’ for variability of pathogens, which is independent of mean pathogen levels. The model is then used to predict minimum depuration times required to achieve norovirus levels which fall within possible risk management levels, as well as predictions of minimum depuration times for other water-borne pathogens found in shellfish. Times for Escherichia coli predicted by the model all fall within the minimum 42 hours required for class B harvest sites, whereas minimum depuration times for norovirus and FRNA+ bacteriophage are substantially longer. Thus this study provides relevant information and tools to assist norovirus risk managers with future control strategies. [ABSTRACT FROM AUTHOR]

The identification of microbial species has depended predominantly upon culture-based techniques. However, the difficulty with which types of organisms are cultured implies that the grown species may be overrepresented by both cultivation and plate counts. In recent years, culture-independent analysis using high-throughput sequencing has been advocated for use as a point-of-care diagnostic tool. Although it offers a rapid and unbiased survey to characterize the pathogens in clinical specimens, its accuracy is reduced by the high level of contamination of human DNA. In this paper, we propose using a culture-independent analysis for a Klebsiella pneumoniae clinical strain within a liver abscess using nanopore sequencing. Owing to the highly-contaminated cell population within a liver abscess, we managed to reduce the confounding effects of human DNA through the use of DNase and differential centrifugation. Genomic DNA was sequenced through the use of Nanopore MinION sequencer and analyzed using a suite of bioinformatics approaches. K. pneumoniae was successfully identified along with antibiotic-resistant genes. Our results indicate that, by integrating real-time nanopore sequencing and bioinformatics software, real-time pathogen identification in a liver abscess can be achieved. [ABSTRACT FROM AUTHOR]

We refine and clinically parameterize a mathematical model of the humoral immune response against Shigella, a diarrheal bacteria that infects 80-165 million people and kills an estimated 600,000 people worldwide each year. Using Latin hypercube sampling and Monte Carlo simulations for parameter estimation, we fit our model to human immune data from two Shigella EcSf2a-2 vaccine trials and a rechallenge study in which antibody and B-cell responses against Shigella′s lipopolysaccharide (LPS) and O-membrane proteins (OMP) were recorded. The clinically grounded model is used to mathematically investigate which key immune mechanisms and bacterial targets confer immunity against Shigella and to predict which humoral immune components should be elicited to create a protective vaccine against Shigella. The model offers insight into why the EcSf2a-2 vaccine had low efficacy and demonstrates that at a group level a humoral immune response induced by EcSf2a-2 vaccine or wild-type challenge against Shigella′s LPS or OMP does not appear sufficient for protection. That is, the model predicts an uncontrolled infection of gut epithelial cells that is present across all best-fit model parameterizations when fit to EcSf2a-2 vaccine or wild-type challenge data. Using sensitivity analysis, we explore which model parameter values must be altered to prevent the destructive epithelial invasion by Shigella bacteria and identify four key parameter groups as potential vaccine targets or immune correlates: 1) the rate that Shigella migrates into the lamina propria or epithelium, 2) the rate that memory B cells (BM) differentiate into antibody-secreting cells (ASC), 3) the rate at which antibodies are produced by activated ASC, and 4) the Shigella-specific BM carrying capacity. This paper underscores the need for a multifaceted approach in ongoing efforts to design an effective Shigella vaccine. [ABSTRACT FROM AUTHOR]

Recent experiments with the bacteria Paenibacillus vortex reveal a remarkable strategy enabling it to cope with antibiotics by cooperating with a different bacterium—Escherichia coli. While P. vortex is a highly effective swarmer, it is sensitive to the antibiotic ampicillin. On the other hand, E. coli can degrade ampicillin but is non-motile when grown on high agar percentages. The two bacterial species form a shared colony in which E. coli is transported by P. vortex and E. coli detoxifies the ampicillin. The paper presents a simplified model, consisting of coupled reaction-diffusion equations, describing the development of ring patterns in the shared colony. Our results demonstrate some of the possible cooperative movement strategies bacteria utilize in order to survive harsh conditions. In addition, we explore the behavior of mixed colonies under new conditions such as antibiotic gradients, synchronization between colonies and possible dynamics of a 3-species system including P. vortex, E. coli and a carbon producing algae that provides nutrients under illuminated, nutrient poor conditions. The derived model was able to simulate an asymmetric relationship between two or three micro-organisms where cooperation is required for survival. Computationally, in order to avoid numerical artifacts due to symmetries within the discretizing grid, the model was solved using a second order Vectorizable Random Lattices method, which is developed as a finite volume scheme on a random grid. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is the most common cause of bacterial gastroenteritis. Major reservoirs are warm-blooded animals, poultry in particular, but Campylobacter can also be transmitted via water. In this paper, we have taken a closer look at the biology and potential virulence of C. jejuni water isolates. Seven C. jejuni isolates from incoming surface water at water plants in Sweden were characterized with whole genome sequencing and phenotypical testing. Multi locus sequence typing analysis revealed that these isolates belonged to groups known to include both common (ST48CC) and uncommon (ST1275CC, ST683, ST793 and ST8853) human pathogens. Further genomic characterization revealed that these isolates had potential for arsenic resistance (due to presence of arsB gene in all isolates), an anaerobic dimethyl sulfoxide oxidoreductase (in three isolates) and lacked the MarR-type transcriptional regulator gene rrpB (in all but one isolate) earlier shown to be involved in better survival under oxidative and aerobic stress. As putative virulence factors were concerned, there were differences between the water isolates in the presence of genes coding for cytolethal distending toxin (cdtABC), Type VI secretion system and sialylated LOS, as well as in biofilm formation. However, all isolates were motile and could adhere to and invade the human HT-29 colon cancer cell line in vitro and induce IL-8 secretion suggesting potential to infect humans. This is, to the best of our knowledge, the first study where C. jejuni water isolates have been characterized using whole genome sequencing and phenotypical assays. We found differences and shared traits among the isolates but also potential to infect humans. [ABSTRACT FROM AUTHOR]

Interactions between microbes are central to the dynamics of microbial communities. Understanding these interactions is essential for the characterization of communities, yet challenging to accomplish in practice. There are limited available tools for characterizing diffusion-mediated, contact-independent microbial interactions. A practical and widely implemented technique in such characterization involves the simultaneous co-culture of distinct bacterial species and subsequent analysis of relative abundance in the total population. However, distinguishing between species can be logistically challenging. In this paper, we present a low-cost, vertical membrane, co-culture plate to quantify contact-independent interactions between distinct bacterial populations in co-culture via real-time optical density measurements. These measurements can be used to facilitate the analysis of the interaction between microbes that are physically separated by a semipermeable membrane yet able to exchange diffusible molecules. We show that diffusion across the membrane occurs at a sufficient rate to enable effective interaction between physically separate cultures. Two bacterial species commonly found in the cystic fibrotic lung, Pseudomonas aeruginosa and Burkholderia cenocepacia, were co-cultured to demonstrate how this plate may be implemented to study microbial interactions. We have demonstrated that this novel co-culture device is able to reliably generate real-time measurements of optical density data that can be used to characterize interactions between microbial species. [ABSTRACT FROM AUTHOR]

This paper forecasts the 2016 canine Anaplasma spp. seroprevalence in the United States from eight climate, geographic and societal factors. The forecast’s construction and an assessment of its performance are described. The forecast is based on a spatial-temporal conditional autoregressive model fitted to over 11 million Anaplasma spp. seroprevalence test results for dogs conducted in the 48 contiguous United States during 2011–2015. The forecast uses county-level data on eight predictive factors, including annual temperature, precipitation, relative humidity, county elevation, forestation coverage, surface water coverage, population density and median household income. Non-static factors are extrapolated into the forthcoming year with various statistical methods. The fitted model and factor extrapolations are used to estimate next year’s regional prevalence. The correlation between the observed and model-estimated county-by-county Anaplasma spp. seroprevalence for the five-year period 2011–2015 is 0.902, demonstrating reasonable model accuracy. The weighted correlation (accounting for different sample sizes) between 2015 observed and forecasted county-by-county Anaplasma spp. seroprevalence is 0.987, exhibiting that the proposed approach can be used to accurately forecast Anaplasma spp. seroprevalence. The forecast presented herein can a priori alert veterinarians to areas expected to see Anaplasma spp. seroprevalence beyond the accepted endemic range. The proposed methods may prove useful for forecasting other diseases. [ABSTRACT FROM AUTHOR]

This is the first study to evaluate the serologic and molecular prevalence of Coxiella burnetii in cattle at national breeding stock farms in South Korea. These government farms have well-organized biosecurity and management systems to prevent livestock diseases. Of the 736 cattle in this study, 77 tested positive for antibodies against C. burnetii antigens (10.5%, 95% CI: 8.3–12.7) and 11 were positive for a C. burnetti infection on PCR analysis (1.5%, 95% CI: 0.6–2.4). Since the 16S rRNA sequences of C. burnetii from all 11 PCR-positive samples were identical, three representative samples (C-CN-3 from the southern region, C-JJ-9 from Jeju Island, and C-CB-37 from the central region) are described in this paper. These three sequences had 99.3–100% identity to those of C. burnetii deposited in GenBank. These sequences clustered with those from USA, Japan, and Greenland, underscoring the sequence similarity among C. burnetii isolates in these countries. Because C. burnetii was detected in cattle at well-managed national breeding stock farms, cattle at non-government operated farms may be more likely to be exposed to C. burnetii in South Korea. Thus, continuous surveillance and control strategies in animals and humans are required to prevent the transmission of C. burnetii to humans. [ABSTRACT FROM AUTHOR]