Characterization of Coastal Urban Watershed Bacterial Communities Leads to Alternative Community-Based Indicators

Background

Microbial communities in aquatic environments are spatially and temporally dynamic due to environmental fluctuations and varied external input sources. A large percentage of the urban watersheds in the United States are affected by fecal pollution, including human pathogens, thus warranting comprehensive monitoring.

Methodology/Principal Findings

Using a high-density microarray (PhyloChip), we examined water column bacterial community DNA extracted from two connecting urban watersheds, elucidating variable and stable bacterial subpopulations over a 3-day period and community composition profiles that were distinct to fecal and non-fecal sources. Two approaches were used for indication of fecal influence. The first approach utilized similarity of 503 operational taxonomic units (OTUs) common to all fecal samples analyzed in this study with the watershed samples as an index of fecal pollution. A majority of the 503 OTUs were found in the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. The second approach incorporated relative richness of 4 bacterial classes (Bacilli, Bacteroidetes, Clostridia and α-proteobacteria) found to have the highest variance in fecal and non-fecal samples. The ratio of these 4 classes (BBC∶A) from the watershed samples demonstrated a trend where bacterial communities from gut and sewage sources had higher ratios than from sources not impacted by fecal material. This trend was also observed in the 124 bacterial communities from previously published and unpublished sequencing or PhyloChip- analyzed studies.

Conclusions/Significance

This study provided a detailed characterization of bacterial community variability during dry weather across a 3-day period in two urban watersheds. The comparative analysis of watershed community composition resulted in alternative community-based indicators that could be useful for assessing ecosystem health.     

A Combined test using both cell sediment and supernatant cell‐free DNA in pleural effusion shows increased sensitivity in detecting activating EGFR mutation in lung cancer patients

Introduction

The aim of this study was to examine whether a combined test using both cell sediment and supernatant cytology cell‐free DNA (ccfDNA) is more useful in detecting EGFR mutation than using cell sediment DNA or supernatant ccfDNA alone in pleural effusion of lung cancer patients.

Methods

A total of 74 lung adenocarcinoma patients with paired samples between primary tumour and corresponding metastatic tumour with both cell sediment and supernatant ccfDNA of pleural effusion cytology were enrolled in this study. Cell sediment and supernatant ccfDNA were analysed separately for EGFR mutations by polymerase chain reaction.

Results

Out of 45 patients with mutant EGFR in primary tumours, EGFR mutations were detected in 23 cell sediments of corresponding metastases (sensitivity; 51.1%) and 20 supernatant ccfDNA corresponding metastases (sensitivity; 44.4%). By contrast, the combined test detected EGFR mutations in 27 corresponding metastases (sensitivity; 60.0%), and had a higher sensitivity than the cell sediment or the supernatant ccfDNA alone (P < .05). Out of 45 patients with mutant EGFR, 24, three and 18 were cytologically diagnosed as positive, atypical or negative, respectively. The detection rate in the combined test was highest (95.8%) in the positive group, and mutant EGFR was also detected in four of 18 samples (22.2%) in the negative group.

Conclusions

A combined test using both cell sediment DNA and supernatant ccfDNA samples increases the concordance rate of EGFR mutations between primary tumour and corresponding metastases. Our findings indicate that supernatant ccfDNA is useful even in cases where the cytological diagnosis is negative.     

Prenatal household size and composition are associated with infant fecal bacterial diversity in Cebu,Philippines

Objectives

The gut microbiome (GM) connects physical and social environments to infant health. Since the infant GM affects immune system development, there is interest in understanding how infants acquire microbes from mothers and other household members.

Materials and Methods

As a part of the Cebu Longitudinal Health and Nutrition Survey (CLHNS), we paired fecal samples (proxy for the GM) collected from infants living in Metro Cebu, Philippines at 2 weeks (N = 39) and 6 months (N = 36) with maternal interviews about prenatal household composition. We hypothesized that relationships between prenatal household size and composition and infant GM bacterial diversity (as measured in fecal samples) would vary by infant age, as well as by household member age and sex. We also hypothesized that infant GM bacterial abundances would differ by prenatal household size and composition.

Results

Data from 16 S rRNA bacterial gene sequencing show that prenatal household size was the most precise estimator of infant GM bacterial diversity, and that the direction of the association between this variable and infant GM bacterial diversity changed between the two time points. The abundances of bacterial families in the infant GM varied by prenatal household variables.

Conclusions

Results highlight the contributions of various household sources to the bacterial diversity of the infant GM, and suggest that prenatal household size is a useful measure for estimating infant GM bacterial diversity in this cohort. Future research should measure the effect of specific sources of household bacterial exposures, including social interactions with caregivers, on the infant GM.

     

Evaluation of fecal mRNA reproducibility via a marginal transformed mixture modeling approach

Background  

Developing and evaluating new technology that enables researchers to recover gene-expression levels of colonic cells from fecal samples could be key to a non-invasive screening tool for early detection of colon cancer. The current study, to the best of our knowledge, is the first to investigate and report the reproducibility of fecal microarray data. Using the intraclass correlation coefficient (ICC) as a measure of reproducibility and the preliminary analysis of fecal and mucosal data, we assessed the reliability of mixture density estimation and the reproducibility of fecal microarray data. Using Monte Carlo-based methods, we explored whether ICC values should be modeled as a beta-mixture or transformed first and fitted with a normal-mixture. We used outcomes from bootstrapped goodness-of-fit tests to determine which approach is less sensitive toward potential violation of distributional assumptions.     

Detection of Helicobacter pylori in stool samples of young children using real‐time polymerase chain reaction

Background

The aims of this study were to develop and validate a multiplex real‐time polymerase chain reaction (q‐PCR) assay of Helicobacter pylori in stool samples of healthy children. Additionally, we determined the prevalence of clarithromycin resistance and cagA gene in H. pylori‐positive samples.

Materials and methods

Archived stool samples from 188 children aged 6‐9 years and 272 samples of 92 infants aged 2‐18 months were tested for H. pylori antigens using enzyme immunoassay (EIA). A multiplex q‐PCR assay was designed to detect H. pylori 16S rRNA and urease and the human RNase P gene as an internal control. Kappa coefficient was calculated to assess the agreement between q‐PCR and EIA.

Results

Laboratory validation of the q‐PCR assay using quantitated H. pylori ATCC 43504 extracted DNA showed S‐shaped amplification curves for all genes; the limit of detection was 1 CFU/reaction. No cross‐reactivity with other bacterial pathogens was noted. Applying the multiplex q‐PCR to DNA extracted from fecal samples showed clear amplification curves for urease gene, but not for 16S rRNA. The prevalence of H. pylori infection was 50% (95% CI 43%‐57%) by q‐PCR (urease cycle threshold <44) vs 59% (95% CI 52%‐66%) by EIA. Kappa coefficient was .80 (P < .001) and .44 (P < .001) for children aged 6‐9 years and 2‐18 months, respectively. Sixteen samples were positive for cagA and three were positive for clarithromycin resistance mutation (A2143G) as confirmed by sequencing.

Conclusions

The developed q‐PCR can be used as a cotechnique to enhance the accuracy of H. pylori detection in epidemiological studies and in clinical settings.     

Diversity,abundance, and possible sources of fecal bacteria in the Yangtze River

The fecal bacteria in natural waters may pose serious risks on human health. Although many source tracking methods have been developed and used to determine the possible sources of the fecal pollution, little is known about the overall diversity and abundance of fecal bacterial community in natural waters. In this study, a method based on fecal bacterial sequence library was introduced to evaluate the fecal bacterial profile in the Yangtze River (Nanjing section). Our results suggested that the Yangtze River water harbors diverse fecal bacteria. Fifty-eight fecal operational taxonomic units (97% identity level) were detected in the Yangtze River water samples and the relative abundance of fecal bacteria in these samples ranged from 0.1 to 8%. It was also found that the relative abundances of the fecal bacteria in locations near to the downstream of wastewater treatment plants were obviously higher than those in other locations. However, the high abundance of fecal bacteria could decrease to the normal level in 2~4 km in the river due to degradation or dilution, and the overall fecal bacteria level changed little when the Yangtze River flew through the Nanjing City. Moreover, the fecal bacteria in the Yangtze River water were found to be highly associated (Spearman rho = 0.804, P < 0.001) with the potential pathogenic bacteria. Collectively, the findings in this study reveal the diversity, abundance, and possible sources of fecal bacteria in the Yangtze River and advance our understandings of the fecal bacteria community in the natural waters.

     

Detection and quantification of 14 <Emphasis Type="Italic">Campylobacter</Emphasis> species in pet dogs reveals an increase in species richness in feces of diarrheic animals

Background  

The genus Campylobacter includes many species, some of which are known human and animal pathogens. Even though studies have repeatedly identified domestic dogs as a risk factor for human campylobacteriosis, our understanding of Campylobacter ecology in this reservoir is limited. Work to date has focused primarily on a limited number of species using culture-based methods. To expand our understanding of Campylobacter ecology in dogs, a collection of fecal samples from 70 healthy and 65 diarrheic pet dogs were examined for the presence and levels of 14 Campylobacter species using quantitative PCR.     

A comparative study of cultural methods for the detection of Salmonella in feed and feed ingredients

Background  

Giardia duodenalis is a ubiquitous protozoan parasite that has emerged as a significant opportunistic human pathogen. G. duodenalis may have a deleterious effect on animal growth and performance, therefore its potential as a production limiting organism should not be discounted. We therefore undertook this study to determine management and environmental factors in feedlots that influence the prevalence and environmental load of G. duodenalis cysts in fecal material deposited by feedlot cattle in the central and western United States.     

Phosphorus and carbohydrate limitation of fecal coliform and fecal enterococcus within tidal creek sediments

Aquatic sediments can be a significant reservoir of bacterial indicators of fecal contamination at levels higher than the waters above them. Several environmental factors have been identified that can enhance the role of sediments as a reservoir for enteric pathogens, including carbon and/or phosphorus availability. In order to investigate the influence of these and other environmental factors on sediment fecal bacteria populations, sediment samples were collected from a coastal watershed in southeastern North Carolina and analyzed for fecal coliform and fecal enterococcus using a modified membrane filtration technique. Measurements of sediment phosphorus, sediment carbohydrate, and environmental factors were made and relationships with bacteria concentrations were assessed. These observations were accompanied by an experimental laboratory manipulation of phosphorus and carbohydrate and their effects on sediment-associated fecal coliform and enterococcus. Field results suggested that sediment-associated indicator bacteria were not limited by sediment phosphorus or carbohydrate. Experimental results suggested that sediment-associated fecal bacteria were more frequently limited by bioavailable carbohydrate. Sediment phosphorus was limiting for fecal enterococcus only where sediment P was initially low (<31 μg P g⁻¹). A strong positive response by sediment fecal coliform concentrations to recent (24 h) precipitation was evidence that stormwater runoff delivers fecal bacteria loadings that are only partly measurable by conventional water sampling schemes, and by driving sediment and sediment P-loading plays a significant role in enhancing aquatic sediments as reservoirs for fecal microbes.     

Distribution and Significance of Fecal Indicator Organisms in the Upper Chesapeake Bay

Total viable aerobic, heterotrophic bacteria, total coliforms, fecal coliforms, and fecal streptococci were enumerated in samples collected at five stations located in the Upper Chesapeake Bay, December 1973 through December 1974. Significant levels of pollution indicator organisms were detected at all of the stations sampled. Highest counts were observed in samples collected at the confluence of the Susquehanna River and the Chesapeake Bay. The indicator organisms examined were observed to be quantitatively distributed independently of temperature and salinity. Counts were not found to be correlated with concentration of suspended sediment. However, significant proportions of both the total viable bacteria (53%) and fecal indicator organisms (>80%) were directly associated with suspended sediments. Correlation coefficients (r) for the indicator organisms examined in this study ranged from r = 0.80 to r = 0.99 for bottom water and suspended sediment, respectively. Prolonged survival of fecal streptococci in most of the sediment samples was observed, with concomitant reduction of the correlation coefficient from r = 0.99, fecal streptococci to total coliforms in water, to r = 0.01, fecal streptococci to fecal coliforms in sediments. The results of this study compared favorably with fecal coliforms: fecal streptococci ratios for the various sample types. Characterization of organisms beyond the confirmed most-probable-number procedure provided good correlation between bacterial indicator groups.     

Identification of canine norovirus in dogs in South Korea

Background

Canine noroviruses (CaNoVs) are classified into genogroups GIV, GVI, and GVII and have been detected in fecal samples from dogs since their first appearance in a dog with enteritis in Italy in 2007. CaNoVs may be a public health concern because pet animals are an integral part of the family and could be a potential reservoir of zoonotic agents. Nonetheless, there was no previous information concerning the epidemiology of CaNoV in South Korea. In the present study, we aimed to detect CaNoV antigens and to investigate serological response against CaNoV in dogs.

Results

In total, 459 fecal samples and 427 sera were collected from small animal clinics and animal shelters housing free-roaming dogs in geographically distinct areas in South Korea. For the detection of CaNoV, RT-PCR was performed using target specific primers, and nucleotide sequences of CaNoV isolates were phylogenetically analyzed. Seroprevalence was performed by ELISA based on P domain protein. CaNoVs were detected in dog fecal samples (14/459, 3.1%) and were phylogenetically classified into the same cluster as previously reported genogroup GIV CaNoVs. Seroprevalence was performed, and 68 (15.9%) of 427 total dog serum samples tested positive for CaNoV IgG antibodies.

Conclusion

This is the first study identifying CaNoV in the South Korean dog population.

     

Assessment of FAE1 polymorphisms in three Brassica species using EcoTILLING and their association with differences in seed erucic acid contents

Background  

FAE1 (fatty acid elongase1) is the key gene in the control of erucic acid synthesis in seeds of Brassica species. Due to oil with low erucic acid (LEA) content is essential for human health and not enough LEA resource could be available, thus new LEA genetic resources are being sought for Brassica breeding. EcoTILLING, a powerful genotyping method, can readily be used to identify polymorphisms in Brassica.     

Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection

Background

In Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study.

Methodology

Stool specimens were collected in a highly endemic area for S. mansoni in Ethiopia and submitted in an investigator-blinded fashion to microscopic examination by Kato-Katz thick smear as well as to real-time PCR using either fresh frozen stool samples or stool smears on fecal cards which have been stored at ambient temperature for up to ten months.

Principal Findings

Out of 55 stool samples, 35 were positive by microscopy, 33 and 32 were positive by PCR of frozen samples and of fecal card samples, respectively. When microscopy was used as diagnostic “gold standard”, the sensitivity of PCR on fresh stool was 94.3% (95%-CI: 86.6; 100) and on fecal cards 91.4% (95%-CI: 82.2; 100).

Conclusions

The use of fecal cards proved to be a simple and useful method for stool collection and prolonged storage prior to PCR based diagnosis of S. mansoni infection. This technique may be a valuable approach for large scale surveillance and post treatment assessments     

Outer membrane protein a of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium activates dendritic cells and enhances Th1 polarization

Background  

Typhoid, which is caused by Salmonella enterica serovar Typhimurium, remains a major health concern worldwide. Multidrug-resistant strains of Salmonella have emerged which exhibit increased survivability and virulence, thus leading to increased morbidity. However, little is known about the protective immune response against this microorganism. The outer membrane protein (Omp)A of bacteria plays an important role in pathogenesis.     

A meta‐analysis of the association between Helicobacter pylori (H. pylori) infection and hyperemesis gravidarum

Background

Hyperemesis gravidarum remains a common, distressing, and significant yet poorly understood disorder during pregnancy. The association between maternal Helicobacter pylori (H. pylori) infection and hyperemesis gravidarum has been increasingly recognized and investigated. This study thus aimed to provide an updated review and meta‐analysis of the topic.

Methods

Using the search terms (H. pyloriOR Helicobacter ORHelicobacter pyloriOR infection) AND (pregnancy OR emesis OR hyperemesis gravidarum OR nausea OR vomiting), a preliminary search on the PubMed, Ovid, Web of Science, Google Scholar, and WanFang database yielded 372 papers published in English between January 1st, 1960 and June 1st, 2017.

Results

A total of 38 cross‐sectional and case‐control studies, with a total of 10 289 patients were eligible for review. Meta‐analysis revealed a significant association between H. pylori infection and hyperemesis gravidarum during pregnancy, with a pooled odds ratio of 1.348 (95% CI: 1.156‐1.539, P < .001). Subgroup analysis found that serologic and stool antigen tests were comparable methods of detecting H. pylori as they yielded similar odds ratios.

Limitations

Although the studies did not have high heterogeneity (I² = 28%), publication bias was observed, and interstudy discrepancies in the diagnostic criteria adopted for hyperemesis gravidarum limit the reliability of findings. Also, 15 of the included studies were from the same country (Turkey), which could limit the generalizability of current findings. The prevalence of H. pylori infection varies throughout the world, and there may also be pathogenic differences as most strains of H. pylori in East Asia carry the cytotoxin‐associated gene A gene.

Conclusion

H. pylori infection was associated with an increased likelihood of hyperemesis gravidarum during pregnancy. Given the high prevalence of H. pylori infections worldwide, detecting H. pylori infection and the eradication of maternal H. pylori infection could be part of maternal hyperemesis gravidarum management. Further confirmation with robust longitudinal studies and mechanistic investigations are needed.     

Distribution and Toxicity Risks of PCBs in the Tidal Flat Ecosystem Near the 37th PCBs Sealed Site on the Coast of Zhejiang Province,China

This study investigated the dioxin-like (DL) PCB concentration and congener distributions in water, sediment, and Boleophthalmus pectinirostris samples collected from a beach near the 37th PCBs sealed site (the hazardous waste storage site was sealed by Zhejiang Province Environmental Monitoring Center) of Zhejiang Province, China, so as to evaluate the harmful effects of PCBs waste sealed sites on the surrounding environment. The results showed that DL-PCBs were in all samples. For all samples, the detection ratio of the mono-ortho congener PCB118 and the non-ortho congener PCB81 reached 100%, and both of their contents correlated well with the total DL-PCBs contents. Levels of low-chloride congeners were higher in environmental samples, while the enrichment of the high-chloride congeners was higher in fish. The content of PCBs in B. pectinirostris was highly related to the sediment environment, thus B. pectinirostris could well indicate environmental PCBs pollution. Considering both of the PCBs content and toxic analysis, even after 4 years of being excavated and cleaned, water, sediment, and fish samples were still at a certain degree of toxicity risks. Some areas were revealed to have a high toxicity risk, suggesting this PCBs sealed site still posed toxicity risks to the surrounding tidal flat ecosystem and might potentially endanger animals and human health.     

Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples

Aims

Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10‐fold serial dilutions of Bacillus anthracis spores using quantitative real‐time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10¹ and 1·3 × 10² CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS).

Methods and Results

The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors.

Conclusions

Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit.

Significance and Impact of the Study

The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples.     

The effect of the macrolide antibiotic tylosin on microbial diversity in the canine small intestine as demonstrated by massive parallel 16S rRNA gene sequencing

Background  

Recent studies have shown that the fecal microbiota is generally resilient to short-term antibiotic administration, but some bacterial taxa may remain depressed for several months. Limited information is available about the effect of antimicrobials on small intestinal microbiota, an important contributor to gastrointestinal health. The antibiotic tylosin is often successfully used for the treatment of chronic diarrhea in dogs, but its exact mode of action and its effect on the intestinal microbiota remain unknown. The aim of this study was to evaluate the effect of tylosin on canine jejunal microbiota. Tylosin was administered at 20 to 22 mg/kg q 24 hr for 14 days to five healthy dogs, each with a pre-existing jejunal fistula. Jejunal brush samples were collected through the fistula on days 0, 14, and 28 (14 days after withdrawal of tylosin). Bacterial diversity was characterized using massive parallel 16S rRNA gene pyrosequencing.     

Associations between duration of first trimester intrauterine exposure to genocide against the Tutsi and health outcomes in adulthood

Objectives

Hundreds of thousands of Rwandans were conceived during the 1994 genocide against the Tutsi, including thousands conceived by genocidal rape. We explore whether the duration of first trimester exposure to the genocide is associated with variation in adult mental health outcomes in individuals exposed to varying degrees of genocide-related stress in utero.

Materials and Methods

We recruited 30 Rwandans conceived via genocidal rape, 31 Rwandans conceived by genocide survivors not raped, and 30 individuals of Rwandan-descent who were conceived outside of Rwanda at the time of the genocide (control group). Individuals were age- and sex-matched across groups. Adult mental health was assessed through standardized questionnaires for vitality, anxiety, and depression.

Results

Among the genocide only group, a longer duration of first trimester prenatal exposure was associated with higher anxiety scores and lower vitality (both p < 0.010), and higher depression scores (p = 0.051). Duration of first trimester exposure was not associated with any measures of mental health among the genocidal rape or control group.

Discussion

Duration of exposure to genocide in the first trimester of gestation was associated with variation in adult mental health among the genocide only group. The lack of association between duration of first trimester exposure to genocide and adult mental health in the genocidal rape group may reflect the fact that stress associated with conception through rape persisted beyond the genocide period itself, encompassing all of gestation and likely beyond. Geopolitical and community interventions are needed in the context of extreme events during pregnancy to mitigate adverse intergenerational outcomes.