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Since Streptococcus pneumoniae transmits through droplet spread, this respiratory tract pathogen may be able to survive in saliva. Here, we show that saliva supports survival of clinically relevant S. pneumoniae strains for more than 24 h in a capsule-independent manner. Moreover, saliva induced growth of S. pneumoniae in growth-permissive conditions, suggesting that S. pneumoniae is well adapted for uptake of nutrients from this bodily fluid. By using Tn-seq, a method for genome-wide negative selection screening, we identified 147 genes potentially required for growth and survival of S. pneumoniae in saliva, among which genes predicted to be involved in cell envelope biosynthesis, cell transport, amino acid metabolism, and stress response predominated. The Tn-seq findings were validated by testing a panel of directed gene deletion mutants for their ability to survive in saliva under two testing conditions: at room temperature without CO2, representing transmission, and at 37°C with CO2, representing in-host carriage. These validation experiments confirmed that the plsX gene and the amiACDEF and aroDEBC operons, involved in respectively fatty acid metabolism, oligopeptide transport, and biosynthesis of aromatic amino acids play an important role in the growth and survival of S. pneumoniae in saliva at 37°C. In conclusion, this study shows that S. pneumoniae is well-adapted for growth and survival in human saliva and provides a genome-wide list of genes potentially involved in adaptation. This notion supports earlier evidence that S. pneumoniae can use human saliva as a vector for transmission.
Citation: Verhagen LM, de Jonge MI, Burghout P, Schraa K, Spagnuolo L, Mennens S, et al. (2014) Genome-Wide Identification of Genes Essential for the Survival of Streptococcus pneumoniae in Human Saliva. PLoS ONE 9(2): e89541. https://doi.org/10.1371/journal.pone.0089541
Editor: Ali Al-Ahmad, University Hospital of the Albert-Ludwigs-University Freiburg, Germany
Received: November 13, 2013; Accepted: January 22, 2014; Published: February 25, 2014
Copyright: © 2014 Verhagen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was financially supported by Nano Cluster of Technology Foundation STW FES0901: FES HTSM, European Community's Seventh Framework Programme FP7/2007-2013 under grant agreement number HEALTH-F3-2009-222983 (Pneumopath project), and Agentschap NL project number OM111009 (Pneumovac project). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Streptococcus pneumoniae is the most common bacterial etiology of community-acquired pneumonia in all ages, and can cause outbreaks in closed settings. The most common manifestations of pneumococcal disease include sinusitis, otitis media, pneumonia and sepsis. The increasing antibiotic resistance and limited serotype coverage of currently available vaccines demonstrates the need for novel approaches in exploring new antimicrobials and vaccines.
All pneumococcal disease begins with the establishment of nasopharyngeal colonization. Once acquired, an individual pneumococcal strain can be carried for weeks to months before its eventual clearance [1]. Pneumococcal carriage induces the production of both mucosal and systemic immunoglobulins. Immunoglobulin G (IgG) and secretory IgA antibodies directed against capsular polysaccharides and surface-associated proteins have been observed in saliva of children in response to colonization with S. pneumoniae [2], [3]. The presence of anti-pneumococcal IgA antibodies in saliva has been associated with a lowered risk of pneumococcal acute otitis media (AOM), while the serum IgG antibody level does not seem to be associated with AOM risk [4]. This suggests that the presence of S. pneumoniae in saliva is associated with the development of local pneumococcal disease.
Transmission of S. pneumoniae, i.e. person-to-person spread, is thought to occur through direct contact with the secretions of colonized individuals. Commonplace activities such as talking, coughing, and sneezing disseminate large amounts of respiratory droplets that subsequently deposit on dry surfaces [5]. These droplets carry bacteria and thus constitute a medium for transmission of S. pneumoniae. Understanding of bacterial survival in droplets is crucial for controlling infection transmission via airborne aerosols and/or large droplet routes. Walsh et al. demonstrated that S. pneumoniae can survive at ambient temperature and humidity for at least four weeks [6]. For this and other studies investigating the importance of fomites and dry surfaces in microbial transmission (summarized in a systematic review by Kramer et al. [7]), bacteria were suspended in Todd-Hewitt broth supplemented with yeast extract (THY), distilled water or saline solution. To our knowledge, the survival of S. pneumoniae in human respiratory secretions has not yet been tested in a laboratory setting. Indirect evidence from studies in humans indicates that S. pneumoniae can survive in saliva and that droplets of saliva may be an important source of transmission of the bacterium. S. pneumoniae was isolated from saliva of patients with stable chronic obstructive pulmonary disease or asthma [8] and recently also from Dutch children between 5 and 10 years of age [9]. Furthermore, in Israeli army recruits frequent sharing of a drinking glass or bottle was an independent risk factor for pneumococcal carriage, suggesting that transmission of pneumococci may occur via saliva [10].
The aim of the current study was to examine the ability of S. pneumoniae to survive and grow in human saliva and to identify the genes essential for its survival in and transmission through saliva in vitro. To this end, we examined the behavior of several encapsulated and unencapsulated S. pneumoniae strains in human saliva under two experimental conditions: at room temperature (RT) without CO2, representing transmission, and at 37°C with CO2, representing in-host carriage. Subsequently, S. pneumoniae genes essential for survival in saliva under these two conditions were identified using the genome-wide negative selection screenings technology Tn-seq [11]. Finally, the roles of individual genes identified by Tn-seq were validated in single and competitive in vitro growth in human saliva.
Results and Discussion
Saliva concentration influences S. pneumoniae growth and survival
In order to test if S. pneumoniae can survive in human saliva, 104 colony forming units (CFU) ml−1 of strain Spain9V-3 (SP195) were incubated with 100%, 50%, 25%, 12.5%, 6.25% and 0% saliva in phosphate-buffered saline (PBS). Viable bacterial counts at t = 0, t = 4 and t = 24 h post-inoculation were determined for two testing conditions: RT without CO2 and 37°C with 5% CO2. At RT without CO2, S. pneumoniae survived in 100% saliva and no significant differences in viable bacterial counts between different saliva concentrations were observed at t = 4 h (not shown). At t = 24 h, the concentration of saliva did significantly affect the survival of S. pneumoniae (p<0.01, Figure 1A left). The number of viable counts was significantly lower in 0% saliva (100% PBS) compared with all other concentrations of saliva (all p-values <0.01). Furthermore, viable bacterial counts in 6.25% and 12.5% saliva were significantly lower than bacterial counts in 25%, 50% or 100% saliva, with p-values varying from <0.01 to 0.040. Viable bacterial counts in 25% saliva did not differ significantly from those in 50% or 100% saliva (Figure 1A left panel).
A. Growth and survival of S. pneumoniae in saliva varies with varying concentrations of saliva. Different dilutions of human saliva in PBS were inoculated with 104 CFU ml−1 S. pneumoniae Spain9V-3 at RT without CO2 (left) or at 37°C with CO2 (right). After 24 hours of incubation, viable bacterial counts of S. pneumoniae were determined and presented as the percentage of the viable bacterial counts detected at t = 0 h. Each point represents a separate replicate. Horizontal lines indicate geometric means. Saliva concentrations for which CFU ml−1 significantly differed from 0% saliva by ANOVA and Tukey post-hoc tests are marked with an asterisk (*). Percentages of saliva for which CFU ml−1 significantly differed from 100% saliva by ANOVA and Tukey post-hoc tests are marked with a black box (▪). B. Effect of bacterial inoculum size on survival and growth of S. pneumoniae Spain9V-3 in saliva. Different number of starting CFU were incubated in 100% saliva at RT without CO2 (left) or at 37°C with CO2 (right) for indicated periods. Data points represent the geometric mean of three replicate experiments. Vertical lines represent the standard deviations of log10 transformed values.
At 37°C with 5% CO2, significant differences in viable bacterial counts between different saliva concentrations were observed at t = 4 h (not shown) and at t = 24 h (Figure 1A right panel). The number of bacteria was significantly lower in 0% 6.25% and 12.5% saliva compared with 25%, 50% and 100% saliva at both time points with p-values varying from <0.01 to 0.049. Interestingly, at 37°C S. pneumoniae showed growth rather than survival in 100%, 50% and 25% saliva with an increase in the number of bacteria at t = 24 h compared with t = 0 h (Figure 1A, right). This suggests that the pneumococcus is able to feed on and grow in human saliva. Growth will lead to large S. pneumoniae cell densities, a condition that may favor transmission of pneumococci through droplet spread.
Because S. pneumoniae survival and growth were optimal in undiluted saliva, all subsequently described experiments were performed with 100% human saliva.
Bacterial inoculum size has no effect on growth and survival of S. pneumoniae in saliva
To examine the influence of inoculum size on survival and/or growth of pneumococci in human saliva, growth characteristics of S. pneumoniae Spain9V-3 were monitored after inoculation of saliva with four different starting inoculae (103, 104, 105 and 106 CFU ml−1). Interestingly, the bacterial inocolum size did not lead to major differences in the growth characteristics of S. pneumoniae in saliva at either growth condition (Figure 1B). At RT without CO2, CFU counts remained near to constant from t = 0 to t = 24 h for all bacterial starting inoculae. For all bacterial starting inoculae growth at 37°C with 5% CO2 was observed in the first 6 h after inoculation. In all cases except for 103 CFU ml−1, there was a subsequent decline in CFU ml−1 between t = 6 and t = 24 h. These results show that at 37°C saliva enables growth of S. pneumoniae until reaching a maximum number of viable bacteria, after which growth of S. pneumoniae diminishes (Figure 1B right). The maximum number of CFU ml−1 was found to be around 106 and was reached faster at higher starting inoculae. A possible explanation for this observation may be that nutrients present in saliva may become scarce as the bacterial population expands until a threshold is reached when not enough nutrients are present for all bacteria to survive.
Growth characteristics of S. pneumoniae in human saliva are strain-specific
The ability to survive and/or grow in saliva was investigated for a selection of S. pneumoniae strains. Globally significant antimicrobial-resistant clones identified by the Pneumococcal Molecular Epidemiology Network (PMEN) with different serotypes were selected [12]. Because we also wanted to assess the role of the capsule for survival of pneumococci in saliva, two unencapsulated strains (R6 and Spain9V-3 Δcps) and two non-typeable strains (AHOY490 and AHOY2831) were included as well (Table 1).
Undiluted human saliva was inoculated with 104 CFU ml−1 S. pneumoniae. All tested S. pneumoniae strains survived in saliva at RT in CO2-poor ambient air and at 37°C in a CO2-rich environment (Figure 2A). At RT without CO2, most S. pneumoniae strains showed survival in saliva with geometric mean viable bacterial counts at t = 24 h around or below 100% of the bacterial starting inoculum (Figure 2A left). At 37°C with 5% CO2 all PMEN strains showed growth with geometric mean viable counts at t = 24 h at least 10-fold higher than the number of bacteria at t = 0 h. The TIGR4 strain showed survival rather than growth at 37°C with 5% CO2 with geometric mean CFU around 10% of the bacterial starting inoculum. The unencapsulated R6 strain also had a geometric mean viable count at t = 24 h around 10% of the bacterial starting inoculum. The unencapsulated derivative of Spain9V-3 (Spain9V-3 Δcps) and the non-typeable AHOY490 and AHOY2831 strains showed growth at 37°C with CO2 with geometric mean viable counts around 250% of the starting inoculum (Figure 2A right).
A. Growth characteristics of several S. pneumoniae strains in human saliva. Strains were incubated in 100% saliva at RT without CO2 (left) or at 37°C with CO2 (right) for 24 h. Viable bacterial counts are presented as the percentage of the viable bacterial counts detected at t = 0 h. Each point represents a single measurement. Horizontal lines indicate geometric means. B. Competitive growth between wild-type Spain9V-3 and its unencapsulated derivative Spain9V-3 Δcps. A 1∶1 ratio of the wild-type and its unencapsulated mutant was inoculated in 100% saliva at 37°C with CO2 and at RT without CO2. Competitive index scores (CI) were determined at t = 4 h and at t = 24 h. Experiments were performed in quadruplicate. Each point represents the log competitive index score of a single measurement. Horizontal lines represent the mean.
We also evaluated the ability of the unencapsulated derivative of Spain9V-3 to survive and grow in saliva when in competition with its wild-type parent. While no difference was seen after 4 h, Spain9V-3 Δcps outcompeted its encapsulated wild type after 24 h at both growth conditions (log CI>0, Figure 2B), demonstrating that the capsule is not essential for the survival in saliva.
Interestingly, all PMEN strains showed much better survival in saliva than the other strains tested at 37°C with 5% CO2, with the exception of EF3030. Since all PMEN strains tested were of the major clonal complex 156 and thus genetically related [13], their similarity in behavior in human saliva may have a genetic basis. The PMEN strains represent a group of important disease-causing pneumococci isolated from patients from different parts of the world [12]. It is tempting to speculate that the wide geographical distribution of the PMEN clones may be related to their increased ability to grow in human saliva and transmit through droplet spread. The epidemiological spread of PMEN clones is especially worrisome because these clones consist of penicillin-resistant and multidrug-resistant pneumococci and thus contribute to the increase in antimicrobial resistance worldwide [12].
Identification of S. pneumoniae Spain9V genes essential for growth and survival in saliva
To identify S. pneumoniae genes essential for growth and survival in saliva, we employed the genome-wide negative selection screen Tn-seq [11], [14]. To this end, a large random S. pneumoniae Spain9V-3 library was challenged by growth at RT in CO2-poor ambient air and at 37°C in 5% CO2. Examination of the relative abundance of each individual transposon mutant in the library before challenge (starting condition) by Tn-seq revealed transposon insertion mutants covered by at least 8 sequence reads in 26,289 unique TA-sites, representing a total of 1615 genes. Tn-seq data can be found on the ESSENTIALS website (http://bamics2.cmbi.ru.nl/websoftware/essentials/links.html).
Our screen identified 78 genes potentially required for survival during 24 h in saliva at RT without CO2, as their corresponding mutants were negatively selected from the mutant-library during challenge (defined as at least 2-fold attenuated survival at 24 h compared with the starting library). After 4 h incubation at 37°C with 5% CO2, mutants of 112 genes were attenuated at least 2-fold. The overlap between genes involved in survival of S. pneumoniae in both conditions was substantial: 55 genes were identified as essential in both conditions (Figure 3A).
A. Overlap between genes identified by Tn-seq as important for survival of S. pneumoniae in saliva at RT without CO2 or at 37°C with CO2. B. Biological properties and molecular functions of genes identified by the Tn-seq screen as essential for pneumococcal growth and/or survival in saliva. The numbers of genes within each category are indicated.
Genes involved in cell envelope biosynthesis are essential for survival of S. pneumoniae in saliva
Functional annotation of the genes selected in either one or both of the challenge conditions (n = 135) revealed that, next to genes with an as yet unknown function, the two most important functional categories were transport and cell envelope biosynthesis (Table S1, Figure 3B). The pneumococcal cell envelope consists of a plasma membrane, the cell wall and the capsule. The cell wall is mainly composed of peptidoglycan (PG) and teichoic acids. Several genes identified as important for the survival of S. pneumoniae in saliva in our study contribute to resistance to antimicrobial peptides (AMPs) and lytic enzymes produced by the host, such as genes belonging to the murMN operon, plsX and pgdA (Table S1). In saliva, positively charged AMPs with membrane-damaging activity and antimicrobial PG lytic enzymes such as lysozyme constitute a first line of defense against infection [15]. Inactivation of murMN or the pgdA gene has been shown to result in hypersensitivity to lysis by lysozyme [16], [17]. We observed that mutants of murMN and pgdA were attenuated in saliva, probably because of their increased sensitivity to lysis by lysozome. Interestingly, pneumococcal cells in which murMN was inactivated completely lost penicillin resistance [18], [19]. This implies that inhibitors of murMN would not only potentially decrease survival of S. pneumoniae in saliva, they may also act as powerful synergists of penicillin against resistant S. pneumoniae strains.
Other attenuated mutants in the functional category of cell envelope biosynthesis included the ortholog of OatA, encoded by SP195_2011 in Spain9V-3 and by SP_2057 in TIGR4. OatA is responsible for O-acetylation of N-acetylmuramyl residues of peptidoglycan, forming N,6-O-diacetylmuramic acid. O-acetylation of the peptidoglycan is the major determinant for lysozyme resistance in Staphylococcus aureus [20] and a similar phenotype was observed for S. pneumoniae and Lactococcus lactis oatA mutants [21], [22]. Interestingly, the regulator Spx, encoded by SP195_A0021 in Spain9V-3 and by SP_0189 in TIGR4, was also found to be attenuated. Spx was found to be responsible for regulating the level of O-acetylation of peptidoglycan in L. lactis [22].
In pneumococci as well as in other gram-positive bacteria, the cell wall envelopes the plasma membrane which consists of phospholipids and various proteins. The plsX gene plays a role in the production of a fatty acid that is involved in membrane phospholipid synthesis in S. pneumoniae and represents a new target for the development of antibacterial therapeutics [23]. Since we observed that the plsX gene was important in survival of S. pneumoniae in human saliva, such new therapeutic agents may also play a role in the prevention of droplet transmission of S. pneumoniae. We hypothesize that the synthesis of fatty acids mediated by plsX is of extra importance for the maintenance of the membrane integrity in a fatty-acid poor environment, such as human saliva [24]. Interestingly, the ortholog of the regulator controlling fatty acid biosynthesis in L. lactis [25], encoded by SP195_0456 in Spain9V-3 and by SP_0416 in TIGR4, was also attenuated, suggesting that this regulator plays a similar role in S. pneumoniae.
Several other genes identified as essential for pneumococcal growth in saliva, such as nagA and glmS, are known key factors involved in cell wall biosynthesis in other bacteria [26], although their roles in S. pneumoniae are not yet fully understood.
Genes involved in transport, stress response and amino acid metabolism are essential for survival of S. pneumoniae in saliva
The largest functional category for which genes were identified by the Tn-seq screening was transport while stress response and amino acid metabolism were respectively third- and fourth-largest (Figure 3B). In these three categories, as well as in the cell envelope biosynthesis category, several operons for which most genes were attenuated were identified, eg, the amiACDEF operon, the mntABC operon, the pst operon and the aroDEBC operon (Table S1). The amiACDEF operon encodes a multicomponent oligopeptide transporter in S. pneumoniae [27]. Oligopeptides are an important source of nutrients, but can also serve as signals for intercellular communication. In a murine model of pneumococcal colonization, deletion of ami genes resulted in pneumococci that were less able to colonize the nasopharynx [28]. While the oligopeptide transport systems are involved in the uptake of nutrients from the external environment, the aro genes are involved in the biosynthesis of aromatic amino acids [29]. Since the concentration of amino acids in saliva is low [15], their biosynthesis is likely to become more important, which explains the observed decrease in survival of the aro mutants in our screen.
The pst operon of S. pneumoniae encodes an ATP-binding cassette phosphate transporter [30]. The uptake of phosphate is of fundamental importance because phosphate is required as a nutrient in the cell physiology of S. pneumoniae. Mutants of pst, unable to transport phosphate over the cell membrane, are less able to take up available phosphate from the saliva which is a likely reason for their underrepresentation in human saliva.
Following these hypotheses concerning the potential roles of plsX, amiACDEF, aroDEBC and pst in pneumococcal survival and/or growth in saliva, we suggest that a limited presence of certain essential nutrients, such as fatty acids, oligopeptides and phosphate, in human saliva can result in a decreased resistance of S. pneumoniae to lytic enzymes when genes coding for nutrient biosynthesis or uptake are inactivated. The exact composition of human saliva varies between individuals and with changes in salivary flow [31]. Anti-pneumococcal antibodies may also be present in human saliva, especially in children [2], [3], and the influence of specific salivary components on the survival and transmission of S. pneumoniae through saliva warrants further investigation.
Manganese is relatively accessible in the human nasopharynx and has been measured at about 36 µM in saliva [32]. The psa operon encodes an ATP-binding cassette transporter for manganese uptake. In pneumococci, manganese transport by this receptor is important for pneumococcal competence and virulence [33], [34]. In addition, psaA mutant strains exhibit increased sensitivity to oxidative stress [35]. Considerable levels of antioxidants are found in human saliva, indicating the presence of oxidative stress in the oral cavity [36]. The uptake of manganese for protection against oxidative stress will thus be important for survival of S. pneumoniae in saliva, which may explain the decreased survival of mutants with a defect mntABC gene in our study. Although the concentration of manganese in saliva is considerable, it is restricted to nanomolar amounts at internal sites [32], [37], [38]. Manganese could thus be an important cue by which the immediate environment is sensed and the transitioning to internal body sites is experienced. Indeed, in clinical S. pneumoniae strains from Chinese patients, psaA expression was significantly higher in blood-derived than in sputum-derived S. pneumoniae [34]. Expression of psaA may thus be involved in invasion of the blood system by S. pneumoniae, making this gene a potentially interesting target for the development of new classes of antimicrobials.
Among the stress response genes, several genes that play a role in the chaperone system were identified, eg, mecA, hslO, tigS and clpB [39]–[42]. In eubacteria, the chaperone system and the chaperones form a functional network, which assists the folding of newly synthesized proteins [43]. Under stress conditions, unfolded and misfolded proteins may be generated, which escape this chaperone network and accumulate as protein aggregates. ClpB, a member of the HSP100/Clp family [44], has the capacity to facilitate the efficient disaggregation and refolding of a large variety of protein aggregates in vitro and in vivo [40], [45]. Possibly, saliva represents a stress condition under which misfolded proteins are generated. ClpB could thus be important for the refolding of protein aggregates, which explains its potential role in the survival of S. pneumoniae in saliva.
Characterization of Tn-seq identified genes that are essential for survival of S. pneumoniae in saliva
For validation of the Tn-seq phenotypes, directed gene deletion mutants were generated in S. pneumoniae Spain9V-3 for representatives from the two main categories: plsX and amiACDEF (belonging to the categories of respectively cell envelope biosynthesis and transport) and for the largest operon for which multiple genes were selected in any of the other functional categories, ie, aroDEBC. Survival of these mutants in saliva under the two challenge conditions was assessed both during single growth, as well as in competition with the wild-type.
In single culture experiments at RT without CO2, the plsX mutant showed a trend towards a decreased survival compared with the wild type at t = 24 (p = 0.056). The amiACDEF and aroDEBC mutant strains did not show significantly decreased survival compared to the wild type at any time point (Figure 4A). After 24 h at 37°C in a CO2-rich environment, all three mutant strains displayed decreased survival compared with the wild- type strain (Figure 4A). However, this was only significant for the aroDEBC mutant (p = 0.032).
A. Single growth experiments. A starting concentration of 106 CFU ml−1 wild-type or mutant bacteria was incubated with saliva at the two conditions. Median and interquartile range (IQR) of the CFU at each time point are displayed. After 24 h at 37°C with CO2, all mutant strains showed decreased survival, but this was only statistically significant for ΔaroDEBC. B. Competitive growth experiments. A 1∶1 ratio of the wild type strain and the mutant strain (5*105 CFU ml−1 for each strain) was inoculated in saliva. Each black point represents the log competitive index score of an individual experiment. Values <0 indicate attenuation of the mutant strain. Horizontal lines represent the mean. After 24 h at 37°C with CO2, the wild-type strain outcompeted all mutant strains, but this was only statistically significant for ΔamiACDEF and ΔplsX.
Competition experiments showed that the wild-type strain outcompeted ΔamiACDEF, ΔaroDEBC and ΔplsX at t = 24 h at 37°C in a CO2-rich environment (Figure 4B) corroborating the Tn seq results, and this was statistically significant for ΔamiACDEF and ΔplsX. At RT without CO2, only the ΔplsX mutant outcompeted the wild-type at t = 24 h with a log CI score of −0.78, but this was not statistically significant. These results are in accordance with the Tn-seq screening, because mutants of the plsX gene showed attenuation at RT without CO2 with a log fold-ratio ≤−1.0, whereas mutants of amiACDEF and aroDEBC did not show attenuation at RT without CO2 (Table S1).
The finding that the competition assays confirmed the Tn-seq results, while no difference was observed between growth of the wild-type compared with the mutants in single culture experiments for plsX and amiACDEF can be explained by the fact that the Tn-seq screening is performed on a mutant library containing a diverse collection of single insertion mutants. The setup of the Tn-seq screening is therefore more comparable to the competition assay than to the single culture assay.
In this study, we demonstrated that various clinically relevant S. pneumoniae strains showed both survival and growth in saliva under conditions representing in-host carriage (37°C with 5% CO2) and transmission (RT without CO2). Additionally, we applied the Tn-seq technology to gain insight into the genetic requirements of pneumococcal growth in saliva, and showed that particularly genes involved in cell envelope biosynthesis, transport, stress response and amino acid metabolism play a role in this process.
The observation that pneumococci survive and grow in human saliva underscores that S. pneumoniae can be transmitted via respiratory droplets. The results described in this study provide a basis for future investigations characterizing the survival and pathogenesis of S. pneumoniae in human saliva.
Ethics statement
Saliva from healthy adult volunteers was collected after obtaining verbal informed consent in the presence of the study team. Written informed consent was not obtained because sampling was non-invasive. The research ethics committee of the Radboud University Medical Centre approved the study protocol and informed consent procedure.
Pneumococcal strains and growth conditions
All pneumococcal strains used in this study are shown in Table 1. S. pneumoniae Spain9V-3 (SP195, PMEN No. 3) was used for the generation of S. pneumoniae transposon mutant libraries since this was a PMEN strain and thus isolated from patients with pneumococcal disease from several parts of the world [12]. Furthermore, the Spain9V-3 strain showed survival after 24 h at RT and growth after 24 h at 37°C with geometric mean viable bacterial counts at t = 24 h around or above 100% of the bacterial starting inoculum (Figure 2). S. pneumoniae strains were routinely grown in Brain Heart Infusion (BHI) broth unless indicated otherwise or on blood agar (BA) plates composed of Columbia agar (Oxoid, Hampshire, United Kingdom) supplemented with 5% defibrinated sheep blood (Biotrading, Mijdrecht, Netherlands). All cultures were incubated in a 5% CO2 incubator at 37°C. S. pneumoniae freezer stocks were prepared from cultures grown to mid-log phase until an optical density at 620 nm (OD620) between 0.2 and 0.3 and stored with 15% glycerol at -80°C. Viable bacterial counts were determined by plating serial 10-fold dilutions in PBS on BA plates. S. pneumoniae mutant libraries and directed mutants were selected with BA plates containing 150 µg ml−1 spectinomycin.
Generation of S. pneumoniae transposon mutant libraries
S. pneumoniae Spain9V-3 mariner transposon mutant libraries were generated essentially as described previously [46]. Briefly, 1 µg of pneumococcal genomic DNA was incubated in the presence of purified Himar1 transposase and 1 µg of plasmid pGSF8 as a donor of the mariner transposon conferring spectinomycin resistance. After repair of the resulting transposition products, 100 ng mutagenized DNA was used for transformation of 100 µl competent S. pneumoniae cells. For mutant libraries, the required number of colonies was scraped from the plates, pooled, grown to mid-log phase and stored in 15% glycerol at −80°C.
Identification of genes essential for S. pneumoniae survival in and transmission through saliva by Tn-seq analysis
For the Tn-seq screen, the S. pneumoniae Spain9V transposon mutant library was first pre-cultured until mid-log phase. Approximately 106 CFU of the mutant library were inoculated into 1 ml saliva, and incubated either at 37°C with 5% CO2 for 4 h or at RT without CO2 for 24 h (n = 4 for each condition). After challenge, samples were supplemented with 15% of glycerol and stored at −80°C. Equivalent CFUs of the recovered libraries and the start library were expanded to mid-log, and chromosomal DNA was isolated using Genomic-tip 20/G columns.
Tn-seq data analysis
The Tn-seq technology was used to profile the relative abundance of each mutant in the library before and after challenge as described previously [14]. For Tn-seq data analysis, FASTQ files were processed via the ESSENTIALS data analysis pipeline (http://bamics2.cmbi.ru.nl/websoftware/essentials/essentials_start.php) [47]. Tn-seq bar code sequences were used to attribute sequence reads to individual samples. Sequence reads were aligned to the Spain9V-3 genome (accession number NZ_ABGE00000000) with a minimum match of 16 nucleotides and collected per unique insertion site (TA dinucleotide sequence) and per gene. The number of unique transposon insertion sites was defined as the positions that had at least 8 "pseudo" sequence reads in the initial "start" library after normalization. In total 26,289 unique mutants where found. Selection criteria for genes of which mutants were affected by the presence of saliva were as follows: (1) a log2 fold-change in sequence reads between the start library and the library challenged with saliva (either for 24 h at RT or for 4 h at 37°C+5%CO2) ≤−1 or ≥1 and a Benjamini-Hochberg adjusted p-value <0.05; (2) a minimum number of reads in the start library of 100; and (3) a fitness score >−3.5, with the fitness score defined as the ratio between actual measured sequence reads and expected sequence reads based on possible TA-insertions sites and library size [47].
Genes were assigned to the functional categories transport, cell envelope biosynthesis, stress response, amino acid metabolism, translation and transcription, carbohydrate metabolism, nucleotide metabolism, cell division and vitamin biosynthesis based on the involvement of their encoded proteins or their orthologs in these processes.
Construction of S. pneumoniae deletion mutants
Directed-deletion mutants of S. pneumoniae were generated by allelic exchange of the target gene with an antibiotic resistance marker as described previously using a megaprimer polymerase chain reaction (PCR) method [46]. In short, an extension PCR was performed to join 400–500 bp 5′and 3′ flanking sequences of the target gene with the spectinomycin resistance cassette (obtained from pR412T7), which was subsequently transformed into naturally competent S. pneumoniae Spain9V-3 bacteria grown in CTM medium. Next, 100 ng of DNA of the first-generation directed mutants was crossed back into the S. pneumoniae Spain9V-3 wild-type strain and selected on spectinomycin plates. At the same time, competent cells were also processed through the transformation procedure without the addition of DNA to obtain a coupled wild-type strain. All primers used in this study (obtained from Biolegio, Nijmegen, The Netherlands) are shown in Table 1.
Deletion of genes from mutants was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) expression analyses. To this end, strains were grown to OD620nm ∼0.5 in THY medium and RNA was isolated as previously described [48]. DNA-free total RNA (500 ng) was reverse transcribed into cDNA using the iScript kit (Bio-Rad). qRT-PCR was performed using SYBR green chemistry on a CFX-96 real-time PCR machine (Bio-Rad). For all mutants, expression of the deleted gene(s) was abolished, while expression levels of the reference gene gyrA were comparable to the wild-type.
Saliva collection
Saliva from healthy volunteers was collected in the early morning after overnight fasting from 11 pm the night before. Oral hygiene was performed not closer than three hours before collection of saliva. After collection, the saliva of at least two donors was pooled, put on ice and centrifuged at 16,000 g and 4°C for 15 minutes. The supernatant was sterilized by ultrafiltration with 0.45 µm filters (Whatman, Dassel, Germany). The sterility of the saliva was verified by incubation of a small portion on BA plates for 48 h at 37°C.
Evaluation of the survival and competitive growth of S. pneumoniae strains in human saliva
For survival assay analyses, a starting concentration of 106 CFU ml−1 bacteria was incubated with saliva at two conditions: 37°C with 5% CO2 and room temperature (RT) without CO2. At t = 0, t = 2, t = 4, t = 6 and t = 24 h samples were taken for CFU count. For competition experiments, a 1∶1 ratio of the wild type strain and the mutant strain (5*105 CFU ml−1 for each strain) was inoculated in saliva. At the same time points as those used for the single cultures, the total number of bacteria was enumerated by plating serial dilutions on blood agar plates and the number of mutant bacteria was enumerated by plating serial dilutions on spectinomycin plates. Experiments were performed in triplo, quadruple or pentuple.
For competition experiments, a competitive index (CI) score was calculated as the output ratio of the viable bacterial counts of mutant to wild type bacteria divided by the input ratio of mutant to wild type bacteria. A log CI score of <0 indicates that the mutant is outcompeted by the wild type. For samples in which no viable bacteria were recovered, the lower limit of detection (222 CFU ml−1) was substituted as the numerator.
Statistical analysis
Statistical differences in growth of S. pneumoniae strains were assessed using unpaired Student's t tests or the nonparametric Mann-Whitney's tests depending on whether or not the log10 transformed values of viable bacterial counts were normally distributed (Kolmogorov-Smirnov's test, p>0.05). Statistical differences in growth of S. pneumoniae in multiple dilutions of saliva were assessed by a one-way analysis of variance (ANOVA) and Tukey post hoc tests. For the competitive growth experiments, a one-sample t test on log-transformed CI scores (with an arbitrary mean of 0 and a p-value of <0.05) was used to calculate statistical significance.
Supporting Information
Table S1.
Genes of S. pneumoniae of which transposon mutants showed attenuated or enhanced survival and/or growth in human saliva, categorized by functional class.
Author Contributions
Conceived and designed the experiments: LMV MIdJ PB PWMH HJB. Performed the experiments: LMV PB KS LS SM MJE CEvdGdJ. Analyzed the data: LMV LS SM AZ HJB. Contributed reagents/materials/analysis tools: PB AZ. Wrote the paper: LMV MIdJ PB AZ PWMH HJB.
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Throughout March, which is Women's History Month, the North Iowa Times will again publish a series of articles highlighting local women. Whether it's through their careers, hobbies, volunteer efforts or unique personalities, these women have become an inspiration to others.
By Audrey Posten, North Iowa Times
Tera Mathis has strived to be physically strong from a young age.
"I lifted in the weight room as a girl, in high school," she recalled. "At the time, it was not a friendly female atmosphere, but I learned a lot from watching the boys."
Marquette, McGregor and other nearby towns along the Mississippi River should brace for the potential of above- or well-above-normal flooding this spring, according to the preliminary hydrologic outlook issued by the National Weather Service (NWS) office in La Crosse on Feb. 21.
In addition to the main-stem Mississippi River, the flooding risk is similar for many tributaries in Wisconsin that feed into it. Western tributaries in southeast Minnesota and northeast Iowa, on the other hand, have a near- to below-normal risk of spring flooding.
The NWS predicts a greater than 95 percent chance the Mississippi will reach the minor flood stage of 16 feet at McGregor this year. Normally, the river has a 52 percent chance of reaching that level in the spring.
Linda Jones, of McGregor, sits with her dog, Toby, who was a gift from her family after her cancer diagnosis 10 years ago. Displayed with her are T-shirts from many years of the Friends Helping Friends fall cancer walk that Linda helps organize. The group's Winter Triathlon and Benefit is March 8 and 9, in McGregor.
Jones shares experiences with personal health crisis and the group she's passionate about
By Becky Ruff
Like many efforts of so many people in communities, both small and large, the group that began what is now known as Friends Helping Friends just started planting seeds where they were needed.
Linda Jones of McGregor was involved when it started.
"I believe it was 1989," she said. Linda remembers there were also a number of people from the community and from local businesses, including the Chamber of Commerce, that started doing fundraisers for various needs.
Edward Carl Davies, 70, of McGregor, passed away on Saturday, Feb. 23, 2019, in Davenport.
He was born on Sept. 19, 1948, in McGregor, to Earl and Elsie (Reinhardt) Davies and attended Mar-Mac High School, graduating in 1966. A life-long lover of water, Ed enjoyed spending as much time as possible on the Mississippi River during his childhood. The majestic power of the river would be an influence on Ed for the rest of his life.
Paul Fiddler, 97, of McGregor, died Friday, Feb. 22, 2019, at Prairie Maison under hospice care in Prairie du Chien, Wis.
He was the son of Michael and Jennie (Albright) Fiddler. Paul was baptized and confirmed Catholic. He attended school in Blue Earth, Minn..
Paul was united in marriage with Delores Radermaker on Aug. 2, 1950, in Butler, Kan. He farmed before being drafted into the United States Army on Dec. 18, 1944. Paul served six months in the South Pacific, before being discharged on Jan. 31, 1946. He was at the signing of Japan Peace Treaty.
Monona resident Kylie Sandstrom completed a mission trip to Haiti earlier this month, where she and other members of the organization HELP Mission International, Inc., helped at an orphanage. Kylie and her husband, Nate, first formed a connection with the orphanage in 2012, and one or both of them have returned every year since. (Photos courtesy of Kylie Sandstrom)
Rebuilding a roof at the orphanage was one of the projects HELP Mission team members worked on when Kylie visited in early February.
These are some of the school desks Kylie helped build.
Kylie Sandstrom, pictured with Pastor John from HELP Mission, as well as Pastor Kelly, who founded the orphanage in Haiti.
Girls at the orphanage receive the baby dolls Kylie brought.
One of the school classes meets.
Kylie Sandstrom poses with other HELP Mission Work Team members during a recent trip to Haiti.
It was 2012. Haiti was still reeling from 2010's devastating 7.0-magnitude earthquake that extensively damaged the Caribbean nation's infrastructure and killed as many as 300,000 people. Living in Fort Dodge then, Nate and Kylie Sandstrom wanted to help.
"We felt like we were called to go after the earthquake," Kylie said. "It felt like God's calling."
After a year of little noticeable activity, chief operating officer Jon Haman said Walz Energy, LLC, is waiting on the weather to begin work again on the 10,000-head cattle feedlot and biogas operation outside Monona.
Work at the site first began nearly two years ago, with plans to construct six open front cattle barns, to go with an additional barn already in existence, as well as a feed storage area, concrete transfer pits and a liquid manure storage lagoon with a capacity of nearly 39 million gallons. Also included on the site will be tanks for anaerobic digestion and methane production.
Haman said the upcoming work will include putting a liner in the lagoon and installing the methane digesters.
A 110-foot section of West Franklin Street, in Monona, will be getting much-needed repairs.
The road narrows at one point, said city administrator Dan Canton at the Feb. 18 council meeting, and drivers are catching the corner and traveling onto the gravel edge, causing the road to deteriorate.
The council approved Bacon Concrete, of Postville, to make the repairs, at a cost of $4,505. Canton said the damaged portion of the road will be excavated out, then replaced with 7-inch-thick concrete. The 110-foot stretch will start out 9 feet wide, then taper down to 4 feet.
Zelma L. Moore, 93, of McGregor, died Wednesday, Feb. 20, 2019, at Crossing Rivers Health in Prairie du Chien, Wis. Zelma Louene "Percie" Moore, the daughter of Percy Roy and Zelma Marie (Huber) Freeman, was born Jan. 1, 1926, in Norwood, Ohio.
She was baptized and confirmed on March 24, 1940, at the Congregational Church in McGregor. She graduated from McGregor High School in 1943 and was the co-valedictorian of her class. Percie was united in marriage with Kenneth James Moore on July 13, 1946, at the Congregational Church in McGregor. Percie worked as an usher at the McGregor Theater, was a telephone operator, worked at Vick's Grocery Store, was an office manager at Brooks-Teaser Insurance and worked at Star Milk Coolers.
MFL MarMac seniors Michael Egan (left), Nathan Johanningmeier and Garret Keehner all placed at last week's state wrestling tournament in Des Moines.
In his last state match, Egan tied the MFL MarMac record for 171 career victories.
Michael Egan earned a fifth-place finish in Class 1A at 145 pounds.
"It means a lot to look back on it, to say you won all these matches, all these big things," said Egan of his time wrestling for MFL MarMac. "It's a testament to the hard work you had to put in. It takes a lot of work to get to where we got this year."
Johanningmeier finished his career with over 150 wins.
Nathan Johanningmeier wrestled into sixth place at 182 pounds in Class 1A.
Johanningmeier felt he wrestled best after his second-round loss. "I felt like I wrestled my best, just coming off that loss and knowing I had to win again to be able to place," he said.
In addition to his superb season record, Keehner also finished with over 140 career victories. He broke MFL MarMac's season record for pins, with 37.
Garret Keehner placed third at 220 pounds in Class 1A.
Keehner went to the tournament a perfect 44-0—the first time any Bulldog has gone to state without a single loss. But he tried to keep that out of his mind as he competed. "I didn't want to jinx myself right away and end up going home with two losses and no medal. So I went in open-minded," Keehner remarked
Three MFL MarMac wrestlers came away from last week's state wrestling tournament in Des Moines with medals. A Class 1A third-place finish for Garret Keehner at 220 pounds, along with fifth for Michael Egan at 145 and sixth for Nathan Johanningmeier at 182, represents the first time since 2010 the school has boasted multiple place winners, estimated head coach Chet Bachman.
"These three kids represented our school very well," he said of the seniors. "I was very proud of them."
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Our NYFW Shopping List
There was diversity in all forms at NYFW and we're happy to see it, here's hoping it continues. Key takeaways: Velvet, Metallics and Vinyl (oh and you can keep your clothes from SS17 too because we're all about repeating).
Outer wear
Raf Simmons made his much awaited return to women's fashion with his first collection for Calvin Klein and everyone in the fashion world is buzzing. This collection is Bauhaus colour blocking dreams.
Looks like we're still doing balloon sleeves
Thom Browne is avant-garde genius.
The pressure was on for Monse's creative directors as they put on two shows at once (they're also creative directors of Oscar de la Renta). While the reviews weren't great, there were some strong pieces in both collections.
Smart-Causal and Casual-Casual
(Because what do these dress codes even mean these days?)
This just makes us feel so warm
Proenza Schouler gladiator sandals
When Hip Hop was hard core and all about the bling bling
Photos Courtesy BOF and Vogue Runway


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Cornish Copper (3.6% ABV, cask)
Plenty of flavour in this very sessionable seasonal beer which is, yes, copper in colour. Easy drinking, it offers a good balance of caramel malt flavours and light berry fruit.
Green Hop (2017) (4.2% ABV, cask)
Fresh, zesty and refreshing — light golden with a white head. The aroma is grassy an slightly spicy. It's light and easy drinking, with some lemon citrus, grassy notes, and light biscuit malt. A gentle bitter finish — really nice. (21-10-2017)
Hops 'n' Honey (4% ABV, cask)
This is the way to do honey beer, Lovely subtlety, light sweetness and a good, grassy hop character. Light honey on the nose. Hopped with Aurora, Celeia and Northdown. (26-03-2016)
Porthleven (4.8% ABV, cask)
Birght golden in colour, citrus lemon notes dominate on this premium bitter. With some sherbet flavour, too, malt comes through later on the palate. Then, there's a really good, long, bitter finish. (02-07-2019)
Pennycomequick (4.5% ABV, cask)
It's that time of the year that a lot of us look forward to down here in Cornwall. The oysters are going on menus, which means that Skinner's Pennycomequick stout is back. As stouts go, it's not the most stout of stouts, but at 4.5% ABV it packs a great flavour punch. I enjoyed my first of the season (the beer is produced twice a year — in autumn and in spring, both ends of the Fal oyster season) at the Old Ale House, Skinner's Truro brewery tap, this week. And it was lovely as ever. It has a smooth, creamy body, reminiscent of a higher-ABV brew, with roast malt notes, of course, but also light coffee, vanilla and Swiss milk chocolate (not our fatty British stuff). There's gentle bitterness on the finish and an aroma of chocolate mousse.


text: Clear your mind and ask your high self (or whatever energy you wish to call upon) to offer you a card for your past, present, and future. You can ask that the spread focus on a particular issue or relationship if you wish. Then, click the image of the card backs below or the button underneath it to reveal your reading.


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H.R.76 - Health Care Commitment Act105th Congress (1997-1998)
BillHide Overview
|Rep. Moran, James P. [D-VA-8] (Introduced 01/07/1997)
|House - National Security; Government Reform
|Latest Action:
|House - 02/03/1997 Executive Comment Requested from DOD. (All Actions)
This bill has the status Introduced
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- Armed Forces and National Security
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Summary: H.R.76 — 105th Congress (1997-1998)All Information (Except Text)
There is one summary for H.R.76. Bill summaries are authored by CRS.
Shown Here:
Introduced in House (01/07/1997)
Health Care Commitment Act - Amends Federal provisions concerning the Civilian Health and Medical Program of the Uniformed Services (CHAMPUS) to direct the Secretary of Defense to enter into an agreement with the Office of Personnel Management (OPM) under which a covered CHAMPUS beneficiary who is also entitled to hospital insurance benefits under Part A of title XVIII (Medicare) of the Social Security Act will be permitted to enroll in a health benefits plan offered through the Federal Employees Health Benefits program in addition to receiving care through a military treatment facility, CHAMPUS, or the TRICARE program. Outlines provisions concerning: (1) required contributions for such coverage; and (2) the management of participants in the plan. Requires the administering Secretaries and the OPM Director to report annually to the Congress describing the provision of health care services to covered beneficiaries under the plan during the preceding fiscal year. Requires the Secretary of Defense to begin to offer the health benefits option described under this Act no later than January 1, 1998.


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This is the third installment related to 24 clues for knowing if you are being a conscious creator during unsettling times. Indeed, during such times, it is important to be "conscious creators" in order to maintain a focus on and to nurture the evolution of human consciousness. It is also fair to say that during unsettling times, each person on the planet, nationally and globally, encounters a variety of choices in this regard to their conscious evolution.
A Way To Exercise Your Personal Power: Vote
By Dr. Carroy (Cuf) Ferguson, Ph.D.
As a psychological and educational association, the Association for Humanistic Psychology (AHP) nurtures a conscious focus on the values of human dignity and humane societies. Currently, the divisive political climate nationally and globally and the lack of sensitivity and civility directed toward our individual and collective human being-ness cry out for a different way "to be."
By Dr. Carroy (Cuf) Ferguson, Ph.D.
AHP President
As human beings on this tiny planet in the cosmos we call Earth, we live as "citizens" in a variety of people-determined land demarcations that we call countries, each with its own unique version of politics. Citizens in the United States and people in the world, therefore, are engaged in a variety of political dramas. It is fair to say that in the United States and globally, the current political climate is very divisive.


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Student Academy Awards 2011: Fifteen Winners Announced at the Samuel Goldwyn TheaterBy Jena Isle on Jun 12, 2011 in Announcement, Entertainment, Movies, Universities •
38th Annual Student Academy Awards Competition Logo
Image credit: oscars.org
Fifteen students were selected as gold medal winners in the 38th Annual Student Academy Awards at the Samuel Goldwyn Theater, June 11, as reported by various entertainment news sites.
The fifteen students, (12 from the US and 3 from other countries) were recipients of the annual competition conducted by the Academy of Motion Picture Arts and Sciences. These young filmmakers' films are available for free screening to the public.
The Annual Student Academy Awards 2011 gold medal winners are:
- "The Vermeers," Tal S. Sharmir, The New School, New York (Gold Medal – Alternative
- "Correspondence," Zach Hyer, Pratt Institute, New York (Gold Medal – Animation)
- "Dragon Boy," Bernardo Warman and Shaofu Zhang, Academy of Art University, California (Gold Medal – Animation)
- "Defective Detective," Avner Geller and Stevie Lewis, Ringling College of Art and Design, Florida (Bronze Medal – Animation)
- "Vera Klement: Blunt Edge," Wonjung Bae, Columbia College Chicago (Gold Medal – Documentary)
- "Imaginary Circumstances," Anthony Weeks, Stanford University (Silver Medal – Documentary)
- "Sin Pais" (Without Country), Theo Rigby, Stanford University (Bronze Medal – Documentary)
- "Thief," Julian Higgins, American Film Institute, California (Gold Medal – Narrative)
- "High Maintenance," Shawn Wines, Columbia University (Silver Medal – Narrative)
- ""Fatakra," Soham Mehta, University of Texas at Austin (Bronze Medal – Narrative)
- "Tuba Atlantic," Hallvar Witzo, The Norwegian Film School, Norway (Gold Medal – Foreign Student Film)
- "Bekas," Karzan Kader, Stockholm Academy of Dramatic Arts, Sweden (Silver Medal – Foreign Student Film)
- "Raju," Max Zaehle, Hamburg Media School, Germany (Bronze Medal – Foreign Student Film)
The complete film clips of the winners of the Student Academy Awards 2011 competition can be viewed at the Oscars.org site.
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In this series of posts, Bobbi Junior brings her unique perspective to bear on the subjects of dementia, and Christian caregiving. Bobbi demonstrates that God is greater than dementia or any other crisis we may face. She shares how being a caregiver is a tiring, challenging and demanding task, and caregiver burnout is a constant threat.
Read Part 6 here.
Bobbi Junior is a contributor to our Christian internet radio station, HopeStreamRadio, through her program entitled "Not Me Lord."
A House Needing Care
Journal Entry, November 6.
Mom's been in her new Assisted Living apartment a little over a week now. She still owns her house, and it's still full of everything she couldn't take with her. We will need to sell it at some point, but not now. Spring will be the logical time to clean it out and put it on the market.
This means hubby, Rick and I will need to keep an eye on the place through this coming winter. I don't like the idea of leaving her house empty, but what choice do we have? I also don't like having to add regular stops to my schedule, to make sure the place is okay over the winter, in addition to visiting Mom at Whyte Hall. Oh, and I still do have that pesky full time job. And a few other commitments as well.
Pity party complete, I turned to Jesus.
Lord, I'm trusting you to set the priorities and make sure we can fit everything in. I will now focus on today, not the winter, not the worry of frozen pipes, or people breaking in, not the task of emptying and selling the house. Just today. That's all I need to deal with for now. Amen.
Snow hasn't settled on the ground yet, so Rick went and raked Mom's lawn today and checked that the windows and doors are locked up and the heat is on low. That should be the last yard work needed until the snow falls, but then shovelling will begin. I'll stop by on Tuesdays and check the house and empty the mail box on my way home from work. Rick will do the same on Fridays. One more thing to add to our schedule.
Processing Grief
I was talking to a friend this weekend and told her how angry and obstinate Mom is being these days. My friend pointed out that in addition to trying to make this transition in the face of all the challenges dementia causes, Mom is probably processing grief at the loss of her house, her home, her independence, her old way of life.
Grief. I hadn't thought of it that way.
It kind of goes both ways, actually. I think my brother, Lawrence and I are grieving as well. When Mom gets on a roll and lets loose with her frustration, and we can't calm her down, we grieve the loss of the intelligent, capable woman she used to be. We still get flashes of that person, but not as much anymore. Some have called this stage of dementia 'the long good-bye'. It's a fitting description.
For me, personally, I find myself trying to figure out what Jesus wants me to feel about my mother, this person I had practically nothing to do with for years, but who's now taking such a prominent place in my life. It's hard to make myself go to see her when I don't know whether I'll be a welcome visitor or seen as her enemy, and on some days, she has me flipping between both roles from one minute to the next.
Logic isn't a tool I can use with her anymore. Loving unconditionally is the command and the lesson, and refusing to meet her anger with my own is the exercise in self-control. I don't always succeed, but I'm trying.
Keeping Joy
Jesus is showing me I need to keep my joy in him no matter what Mom says or does. My task these days is to confidently, in Christ, confront what needs to be confronted when it's in Mom's best interest, and not let it change me or my attitude in a negative way. Above all, I'm to remain respectful of her and help her maintain her dignity.
That's quite the job description, and I don't have a chance of achieving it on my own. This came through loud and clear in church this morning. Pastor Brad talked about the story in Numbers Ch 20 where God instructed Moses to tell the rock to yield its water for the people to drink. Instead of speaking to the rock, in verse 10 Moses says, "Hear now, you rebels: shall we bring water for you out of this rock?" And Moses lifted up his arm and struck the rock with his staff.
Poor Moses. Clearly, the man had lost his patience. He was working hard to do the Lord's bidding and those doggone people were still grumbling and complaining. Moses sort of did as the Lord said regarding the need for water, but instead of having an attitude of obedience, it was more like, "I'll show them".
I get it, Moses! I'm in the same boat. I want to MAKE Mom feel better, more lucid, relaxed, and I'm doing everything I can to achieve that, but she's still miserable and angry. God said Moses wasn't trusting Him to deal with the murmuring Hebrew nation. I guess I'm not always trusting God to meet Mom's needs either.
It's hard, though. It feels like I'm shirking my responsibility, not being with Mom every minute, not finding ways to keep her safe, not being able to make her think clearly.
Feeling Sorry For Myself
I guess I was feeling kind of sorry for myself. As Pastor Brad continued with the sermon, I brought my slightly self-righteous frustration to the Lord, and finally I opened my ears wide enough to hear him. It was very clear in my spirit as Jesus said, "Stop it. Your mother is MY responsibility. If you believe in me, if you trust me with your life, why don't you believe that I'll care for your Mom and her life as well?"
For a moment I was taken aback, but then I remembered another teaching from the Lord. Jesus asks that I give a cup of water, but not that I force the water down the person's throat. He asks that I visit someone in prison, but it's not my job to make them happy about their circumstance. He tells me to give a coat to someone who's naked, but He doesn't say I'm to force them to wear the clothes or be thankful for them.
As we sung the closing hymn, I came to a solid conviction.
My job is to serve according to the need, and do it as it says in Colossians 3:23, Whatever you do, do your work heartily, as for the Lord rather than for men. I'm serving Jesus, not Mom. How she responds is her choice, and not my responsibility. Through it all, I need to keep asking myself: Do I have total trust and confidence in the Lord, believing he'll carry out what needs to happen? And if I do, am I acting as though I do? That's the piece I need to pay attention to!
Read Part 1 here.
Bobbi Junior
Read and hear more from Bobbi Junior on the contributor's page. You can also find Bobbi at her website, The Reluctant Caregiver, at bobbijunior.com
Bobbi's program, "Not Me Lord" airs on HopeStreamRadio.
Contact Info
If you have enjoyed reading this post and wish to send us a comment or share a prayer request, please don't hesitate to contact us and let us know.
Raking Leaves – Julia Freeman – Woolport
Church – Joe Jessop


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PM Saturday - Kanban Essentials Improving How We Work
Alan Zucker, PMP, ACP
PMIWDC Non-Member Rate
This page is from a past PMIWDC event.
If this is not what you were looking for, visit the PM Saturday page to find out about upcoming events, or read more about why PMIWDC does not delete these pages.
Cask LLC
475 Aquia Towne Center Drive - Suite 305
Stafford, VA, 22554
Registration: 08:15 a.m. – 09:00 a.m.
Breakfast buffet and network with project management professionals: 08:30 a.m. – 09:00 a.m.
Announcements: 08:45 a.m. – 09:00 a.m.
Featured presentation: 09:00 a.m. - 10:00 a.m.
Wrap up: 10:00 a.m. – 10:15 a.m.
Pre-registration deadline is October 14th
Member Pre-registration: $15
Non-Member Pre-registration: $20
On-Site Member Rate: $20 (cash or check only)
On-Site Non-Member Rate: $25 (cash or check only)
Cancellations and refund requests are accepted up to 48 hours prior to the day of the event. We cannot offer refunds requested after that time.
About the Program
Agile practices are sweeping through the project management industry. Agile is being used in industries as varied as software development to organizational leadership and news reporting. Agile projects are 4-times more likely to be successful than traditional waterfall projects, according to the Standish Group.
Lean and Kanban practices are at the heart of many Agile practices. These practices are most often associated with Toyota's manufacturing model. However, they are broadly applicable to project management.
In "Kanban Essentials: Improving How We Work," Alan Zucker demonstrates how we can better manage our projects by using these techniques. Participants will: Participate in a hands-on exercise that demonstrates reducing the amount of work going through the system actually increases output and value; Recognize the 8-types of waste that are part of our lives as knowledge workers; and Learn the basics to set-up and maintain a Kanban board, as well as conduct an effective daily stand-up.
These practices will improve how you manage your projects work. Implementing the Kanban board and daily-ups will:
Improve transparency to the team's work and its status;
Increase individual accountability to assigned tasks; and
Promote collaboration among team members.
Free parking will be available.
Industry / SectorGovernment
Federal, state, local or tribal government entities / Contractor for such entitiesI.T.
Information Technology, including organizations that utilize Agile development methodologies
About the Speaker
Alan Zucker, PMP, ACP
Alan Zucker was practicing Agile techniques well before the term was coined. In the early 1990s he employed the agile principles of collaboration, minimal documentation, to deliver a mission critical, mainframe application for the US Treasury. His agile skills were further honed during his years in the fast paced and dynamic telecommunications industry. He also played a leadership role in the Agile transformation of a major financial institution; during this period, the number of annual production releases increased 4-fold, average release durations declined by two-thirds, and project management execution costs were cut in half.
In 2016, he founded Project Management Essentials to provide training and advisory services. He provides Agile, DevOps, and traditional project management training. He also works closely with his clients as they transform their practices.
Alan received his undergraduate degree from The George Washington University and a Master's Degree in Economics from the University of Maryland. He holds a Masters Certificate in IT Project Management from the George Washington University and is a certified Project Management Professional (PMP) and Agile Professional (ACP) by the Project Management Institute (PMI). He also holds Agile certifications as a Scrum Master (CSM) and Scale Agilist (SAFe 4.0 SA).
About the PM Saturday
The PM Saturday quarterly breakfast meeting continues with an engaging speaker, a delightful breakfast, and opportunities to connect with professionals located in the southern-most region of Northern Virginia. Come join us and discover that Saturday morning is also a great time to earn PDUs and to network!


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If you've not yet had chance to finish the Moon Stealer series, the final dramatic book is available FREE on Kindle until 10th July – GRAB A COPY WHILE YOU CAN!
How to Draw: 3 x Videos
It's always interesting to see how other people work. Dylan Gibson, the illustrator for my latest book, The Curious Disappearance of Professor Brown, has filmed three short videos showing you how he draws some of the characters from the book.
The Children of the Light
The fourth and final book in the Moon Stealer series is now available Read More
Round up of the week so far
This week Mr Lawrence Pinkley, the reluctant detective from The Curious Disappearance of Professor Brown, is on the move again. On Monday 2nd December I wrote a guest post on Jennifer Loiske's blog about why I find writing for young people more liberating than writing for adults. Today, Tuesday 3rd December, Carol Bond's blog has… Read More


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Recycling Greywater in Toilets
By dgaudio on December 19, 2012
Water conservation is an important part of energy efficiency. Though it seems as though water is plentiful in our society it is actually a great burden on our energy supply to treat and clean this water for everyday use in our plumbing system. We conserve water not because we fear that the water will run […]
[Read More]
Posted in Plumbing, Save Green | Tagged conservation, conserve, energy, greywater, reduce, toilet, water | Leave a response
Why You Should Conserve Water and 5 Ways to Do It!
By dgaudio on December 5, 2012
We as Americans tend to take our home plumbing systems for granted. Water is cheap and seemingly abundant. However, we can always cut back and there are plenty of reasons to do so. Why You Should Conserve Water Save Money: You pay for every drop of water that comes through your home. Save a little […]
[Read More]
Posted in Plumbing | Tagged conserve, efficiency, energy, lawn, shower, waste treatment, water | Leave a response


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– WWE posted this video of Rey Mysterio driving the pace car at the recent NASCAR Auto Club 400 race. The event took place on Sunday, March 17 from the Auto Club Speedway in Fontana, California.
– The series of live WWE Now videos for WrestleMania 35 Week kicked off earlier today with Seth Rollins speaking to Cathy Kelley on the WWE digital platforms. Thursday will feature a "Then, Now and Forever" discussion with WWE Hall of Famer Shawn Michaels, Johnny Gargano and Kofi Kingston, while there will be a roundtable discussion on Friday with WWE UK Champion Pete Dunne, Ali and Bianca Belair. WWE Women's Tag Team Champions Sasha Banks and Bayley will talk with RAW Tag Team Champions The Revival on Saturday, and the streams will end on Sunday with a live WrestleMania 35 preview featuring WWE Hall of Famer Kurt Angle, Rowan, WWE Champion Daniel Bryan and WrestleMania 35 host Alexa Bliss.
Below is WWE's announcement on the streams:
WWE Now streams daily during WrestleMania 35 Week
WWE Now will head to the New York–New Jersey area for its fourth year of coverage of WrestleMania Week with exclusive interviews featuring Superstars from Raw, SmackDown LIVE and some surprise guests.
The shows will stream on WWE's official Facebook and Twitter pages and YouTube channel, starting Wednesday at noon with an in-depth interview with Seth Rollins.
Schedule (subject to change):
* Seth Rollins reflects on his road back to the World Championship picture on Facebook, Twitter, YouTube
* Shawn Michaels, Kofi Kingston and Johnny Gargano talk about the state of WWE "Then, Now and Forever" — Thursday, noon ET
* Ali, WWE United Kingdom Champion Pete Dunne and Bianca Belair sit down for a roundtable discussion about the future of WrestleMania — Friday, noon ET
* The Boss 'N' Hug Connection and The Revival discuss the state of tag team competition in WWE — Saturday, noon ET
* Live WrestleMania preview featuring WWE Champion "The New" Daniel Bryan with Rowan, Kurt Angle, WrestleMania host Alexa Bliss and more — Sunday, 1 p.m. ET
– Triple H and Stephanie McMahon were featured guests at the 18th Annual CAA World Congress of Sports in Dana Point, California on Wednesday. The event was sponsored by Sports Business Journal, who previously brought Stephanie to their panel at the 2018 Game Changers Conference. Stephanie tweeted the following photo from the event:
Had a great time speaking at the CAA World Congress of Sports with @TripleH and Abe Madkour of @sbjsbd about #WrestleMania, the #WomensEvolution, the importance of hiring talent with passion and so much more. Thank you for having us! On our way back to NYC! #4DaysToGo #SBJWCS pic.twitter.com/ZY8WaBzdRz
— Stephanie McMahon (@StephMcMahon) April 3, 2019
Follow Marc on Twitter at @this_is_marc. Send any news, tips or corrections to us by clicking here.


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TOKYO -- When Akio Toyoda took over as president of Toyota Motor Corp. in the summer of 2009, he likened his challenging mission to "setting sail in a storm."
Little did he know how choppy the seas would be.
At the time, he was grappling with Toyota's first operating loss in 70 years. Bad enough. But each year, his fledgling administration has been buffeted by unprecedented crisis after crisis.
In 2010, his company was assailed by a global recall meltdown affecting millions of vehicles as Toyota battled accusations of unintended acceleration glitches and other quality lapses.


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Pregnant Porsha Williams Is Glowing in Her First "Real" Maternity Dress
The Real Housewives of Atlanta mom-to-be is expanding her pregnancy style.
After months of styling her baby bump in dresses and crop tops from her own pre-pregnancy wardrobe, Porsha Williams has decided to expand her pregnancy style. The Real Housewives of Atlanta mom-to-be is now venturing through the racks of maternity fashion.
Now, we know what you're thinking. Yes, maternity wear doesn't exactly have the best rep for being chic, but (thankfully) maternity brands have come a long way recently, and Porsha's latest look is proof. Earlier this week, the radio host and actress took to Instagram to show off her first piece of designated maternitywear: a curve-hugging striped dress, which is super cute if you ask us. It even resembles a bodycon dress Porsha wore back in October.
Today I wore my first "real" Maternity dress! Dennis couldn't wait to take me maternity shopping for traditional mommy clothes," Porsha wrote.
The RHOA stunner also shared one of her pregnancy fashion hacks: "PS: I'm wearing my belly support belt," she shared.
The Lookbook is Bravo's celebration of personal style. We keep you up to date on the inspiring looks from trend-setting stylistas, test and report on the most-coveted fashion and beauty products, and offer solutions to all your style-related conundrums. Like us on Facebook to stay connected to our daily updates.


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How to Add Contacts to Your HTC One on the Fly
When you get a call, a text, or an email from someone who isn't in your Contacts list, you're given the option to create a profile for that person. The same is true when you initiate contact with someone who isn't in your Contacts list. You can add contacts on the fly:
1Tap the phone icon.
When you first access the Phone application, it brings up the keypad with a blank screen, as seen in the left screen.
2Start dialing the number.
When you start entering the first number, a pop-up menu asks whether you want to add to your contacts in the People application.
Be patient. As you continue to type, your phone tries to guess whose name you're typing. The digits you have typed are included in the phone number for your good buddy, Robert Baden-Powell. As a courtesy, it tries to offer you a chance to save your tapping finger and just call Robert.
3If the first guess is not correct, to call someone else besides Robert, tap the 2 and see the possible contacts.
If Robert isn't the right person to call, you can see that there are two people with that number sequence in your Contacts list. However, if you're dialing a new number for the first time, just keep on typing. Your phone will take those digits until it no longer recognizes the number. Eventually it gives up on guessing.
4When done typing the number, tap Save to People.
Choose whether this is a new contact or a new number for an existing contact. This brings up the option to save it as a new contact or to add this phone number to an existing contact.
5Tap Create Contact.
An empty contact profile will give you the option to add a name. Enter as much information on this contact as you want.
6Tap Save at the top of the screen.
This contact is soon synced with your Gmail account.


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Visitors to Chongqing will find that the Hyatt Regency Chongqing Hotel is a fantastic accommodation choice. From the property, it's easy to reach the Jiangbei Area, just 3km away.
Boasting a convenient location, the hotel is just 5km from Chongqing North Railway Station and 22km from Jiangbei International Airport. Being just minutes away from Guanyinqiao Metro Station allows guests to explore the city with ease.
This Hyatt Regency Chongqing Hotel makes a great place to kick back and relax after a long day of sightseeing. Airport pickup can be arranged by the Hyatt Regency Chongqing Hotel upon request.
This Hyatt Regency Chongqing Hotel provides parking on site.Germaphobes can rest easy - the level of cleanliness is highly rated at this hotel.
This Hyatt Regency Chongqing Hotel is the usual choice for a large number of business travelers. This hotel is one of the most popular choices in Chongqing.
Hotel Adress:
No. 11 Beicheng Tian Street, Jiangbei District, Chongqing, China
Hotel Reservation Telephone:
008620-8600 9099 (CouponCode:12357) (Busy or no answer, online booking please!)
Hotel Front Desk Telephone:
008623-67751234 (Catering, conference,sauna,entertainment, Invoice, traffic)


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Odell Beckham Jr. being animated isn't exactly news, but it was harped on pretty regularly Thursday night.
The New York Giants squared off against the Philadelphia Eagles in "Thursday Night Football." Beckham didn't light up the stat sheet, but he did make waves for being pretty demonstrative on the sideline — namely bodying a massive fan.
The broadcast of the game on FOX kept harping on Beckham for a variety reasons, often in a less-than-flattering light. So much so that it eventually drew the ire of retired NFL tight end Martellus Bennett.
Well into the second half, Bennett shredded the broadcasters in a very NSFW tweet for taking aim at Beckham.
Again, this tweet has some pretty strong language, so be forewarned.
Bennett didn't stop there.
Not mincing words.
The Giants have been a mess this season, and while Beckham hasn't been his best, there are multiple things wrong on both sides of the ball, like Bennett points out.
So while bringing up Beckham is bound to happen since he's a hot-button player, it appears some are finding it's reached extreme degrees.
Thumbnail photo via Brad Penner/USA TODAY Sports Images


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[As usual, dots between square brackets indicate cuts made by Sidney Colvin. For full, correct and critical edition of this letter, see Mehew 4, 1176.]
To Edmund Gosse [Colvin 1911, 2, pp. 155-156]
[Hyères, November 1883]
My dear Gosse,
I have now leisurely read your volume; pretty soon, by the way, you will receive one of mine.
It is a pleasant, instructive, and scholarly volume. The three best being, quite out of sight – Crashaw, Otway, and Etherege. They are excellent; I hesitate between them; but perhaps Crashaw is the most brilliant.
Your Webster is not my Webster; nor your Herrick my Herrick.
On these matters we must fire a gun to leeward, show our colours, and go by. Argument is impossible. They are two of my favourite authors: Herrick above all: I suppose they are two of yours. Well, Janus-like, they do behold us two with diverse countenances, few features are common to these different avatars;
and we can but agree to differ, but still with gratitude to our entertainers, like two guests at the same dinner, one of whom takes clear and one white soup.
By my way of thinking, neither of us need be wrong.
The other papers are all interesting, adequate, clear, and with a pleasant spice of the romantic. It is a book you may be well pleased to have so finished, and will do you much good. The Crashaw is capital: capital; I like the taste of it. Preface clean and dignified.
The handling throughout workmanlike, with some four or five touches of preciosity, which I regret.
With my thanks for information, entertainment, and a pleasurable envy here and there. – Yours affectionately,


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Upcoming Showsno upcoming shows listed
Past Shows
Sat. July 9th 2005Linda Villareal
Sat. August 14th 2004Linda Villareal
Sat. August 7th 2004Linda Villareal
View All Past (16)
Wed. July 21st 2004
@ Myles of Beans (All Ages)Linda Villareal
Linda Villareal
Unknown - Confirmed: Mar. 12, 2007 (Awaiting Update)
from Vancouver BC
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Status: Unknown
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The 5 Greatest Mistakes Students Make When Selecting a university
Okay, you're a senior contributing to to affect colleges, however you may well ask , which colleges? Some students begin thinking about colleges their sophomore year, however, many students steer obvious from the subject prior to the eleventh hour. Remember, it is rarely way too late to develop a great college list.
Unless of course obviously you're a glutton for punishment, you will probably affect no more than 10 or 12 colleges. How are things likely to generate that list? Which colleges suit you're just like a person and student? How will you steer obvious from the five finest mistakes students make when deciding on colleges? They are:
1) Not doing any research on yourself
The beginning point a college search process is working out what you're and also the factor you'll need in the college. What type of college do you want to attend? Big, medium-sized or small? Can it be essential that you should conserve a specific kind of setting, e.g., in the city, college town or province? Do you want to maintain an educationally demanding atmosphere then one that's laid-back? What type of students do you want to be for sale? Individuals as if you or possibly a really diverse group, including worldwide students?
2) Not doing research about colleges
How can we get quality information regarding colleges? There are a number of how. First, there is a superb college guidebooks such as the Fiske Self-help guide to Colleges, The Insider's Self-help guide to the colleges and universities That Make A Difference that provide information regarding colleges in the student's perspective. You might consult with people you trust–parents, your counselor, teachers, and students from your secondary school who're now at different colleges–about which colleges they recommend. Attending different college fairs where admissions representatives come to discuss their colleges is definitely an especially good source. And, clearly, you'll find online sources such as the Internet college searches. From your research, consider a listing check out just what the GPA and test scores stats are appropriate for that universities and the way they match yours.
3) Being too scared to consider a danger
Sometimes students underestimate their chances for college acceptance, particularly when they have a learning disability. Understand that colleges are very supportive to students who've learning issues and sometimes are forgiving of under stellar grades and test scores. Students who're gifted athletes and/and also have special talents sometimes do not understand how interested colleges might be included. Be smart in assembling your college list by utilizing to a lot of us of Achieve schools (where there is a 25% chance of being recognized), Pretty Good Chance schools (where there is a 50% or better chance of being recognized) and assured factor schools (where there is a 75% or better chance of stepping into). An excellent college list includes a number of college choices, but everyone needs to be a university that you might want to visit. Finally, some students are frightened to get using their buddies, family or possibly their hometown. Most students usually undergo some form of homesickness in their newcomer year. The higher you do your research about colleges, the less chance there's of homesickness like a real issue.
4) Being too arrogant
Some students who've stellar academic records and effective test scores confine themselves to joining numerous most likely probably the most selective colleges. They leave Pretty Good Chance and assured Factor schools business lists. This is often a big mistake and a kind of admissions arrogance or naïveté. There are lots of documented times when 4.5 GPA/2340 test score applicants are switched lower by colleges. Universities and colleges are very idiosyncratic with regards to whom they choose. Therefore, it is crucial that you just select numerous schools that offer the choices you need, including some that will not be highly selective. The greater serious result's to locate yourself not proven to the universities that you just placed on since these were Reaches. With meticulous planning, without to occur.
5) Being too Lazy
Joining colleges nowadays is tough. It takes a lot of research to discover the way in which your individual characteristics and individual colleges match. After you have an excellent college list, you need to spend time finishing the applications in a fashion that maximizes your options. Keep in mind that college applications which are only a little better and quite different when compared with competition's are the types that finish off is the best.
Finding which colleges to make use of to can be displayed being an overwhelming task. By remaining from the 5 finest mistakes you'll be able to increase the probability of getting many colleges adore you.


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Phone: (903)262-1920
Degrees and Certifications:
Texas Tech University B.A. in Exercise Science/minor in secondary education Certifications: Generalist EC-6, Physical Education EC-12
Mr. Ricardo Vela
5th & 4th Grade Math
Conference M-F: 12:50-1:40 pm
Room D225
I have been an educator since 2012 but this is my second year at Bonner Elementary. I believe every student has the potential to be successful. My goal is to help my students meet their goals through consistency, hard work, and dedication. It is not only a goal for my students to learn academics in the classroom, but life skills to be successful outside of the classroom. Please feel free to contact me, best method is email. Let me know how I can best help to meet our student's needs.