Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys.

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Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons

Summary
The accelerated growth of finfish aquaculture has resulted in a series of developments detrimental to the environment and human health. The latter is illustrated by the widespread and unrestricted use of prophylactic antibiotics in this industry, especially in developing countries, to forestall bacterial infections resulting from sanitary shortcomings in fish rearing. The use of a wide variety of antibiotics in large amounts, including non-biodegradable antibiotics useful in human medicine, ensures that they remain in the aquatic environment, exerting their selective pressure for long periods of time. This process has resulted in the emergence of antibiotic-resistant bacteria in aquaculture environments, in the increase of antibiotic resistance in fish pathogens, in the transfer of these resistance determinants to bacteria of land animals and to human pathogens, and in alterations of the bacterial flora both in sediments and in the water column. The use of large amounts of antibiotics that have to be mixed with fish food also creates problems for industrial health and increases the opportunities for the presence of residual antibiotics in fish meat and fish products. Thus, it appears that global efforts are needed to promote more judicious use of prophylactic antibiotics in aquaculture as accumulating evidence indicates that unrestricted use is detrimental to fish, terrestrial animals, and human health and the environment.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1462-2920.2006.01054.x

Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment

To estimate the global illness and deaths caused by rotavirus disease, we reviewed studies published from 1986 to 2000 on deaths caused by diarrhea and on rotavirus infections in children. We assessed rotavirus-associated illness in three clinical settings (mild cases requiring home care alone, moderate cases requiring a clinic visit, and severe cases requiring hospitalization) and death rates in countries in different World Bank income groups. Each year, rotavirus causes approximately 111 million episodes of gastroenteritis requiring only home care, 25 million clinic visits, 2 million hospitalizations, and 352,000–592,000 deaths (median, 440,000 deaths) in children <5 years of age. By age 5, nearly every child will have an episode of rotavirus gastroenteritis, 1 in 5 will visit a clinic, 1 in 60 will be hospitalized, and approximately 1 in 293 will die. Children in the poorest countries account for 82% of rotavirus deaths. The tremendous incidence of rotavirus disease underscores the urgent need for interventions, such as vaccines, to prevent childhood deaths in developing nations.

/pdf/global-illness-and-deaths-caused-by-rotavirus-disease-in-4r3lieayv1.pdf

Global Illness and Deaths Caused by Rotavirus Disease in Children

Few studies provide data on the global morbidity and mortality caused by infection with Shigella spp.; such estimates are needed, however, to plan strategies of prevention and treatment. Here we report the results of a review of the literature published between 1966 and 1997 on Shigella infection. The data obtained permit calculation of the number of cases of Shigella infection and the associated mortality occurring worldwide each year, by age, and (as a proxy for disease severity) by clinical category, i.e. mild cases remaining at home, moderate cases requiring outpatient care, and severe cases demanding hospitalization. A sensitivity analysis was performed to estimate the high and low range of morbid and fatal cases in each category. Finally, the frequency distribution of Shigella infection, by serogroup and serotype and by region of the world, was determined. The annual number of Shigella episodes throughout the world was estimated to be 164.7 million, of which 163.2 million were in developing countries (with 1.1 million deaths) and 1.5 million in industrialized countries. A total of 69% of all episodes and 61% of all deaths attributable to shigellosis involved children under 5 years of age. The median percentages of isolates of S. flexneri, S. sonnei, S. boydii, and S. dysenteriae were, respectively, 60%, 15%, 6%, and 6% (30% of S. dysenteriae cases were type 1) in developing countries; and 16%, 77%, 2%, and 1% in industrialized countries. In developing countries, the predominant serotype of S. flexneri is 2a, followed by 1b, 3a, 4a, and 6. In industrialized countries, most isolates are S. flexneri 2a or other unspecified type 2 strains. Shigellosis, which continues to have an important global impact, cannot be adequately controlled with the existing prevention and treatment measures. Innovative strategies, including development of vaccines against the most common serotypes, could provide substantial benefits.

/pdf/global-burden-of-shigella-infections-implications-for-5g7avzciiu.pdf

Global Burden of Shigella Infections: Implications for Vaccine Development and Implementation of Control Strategies

ETEC is an underrecognized but extremely important cause of diarrhea in the developing world where there is inadequate clean water and poor sanitation. It is the most frequent bacterial cause of diarrhea in children and adults living in these areas and also the most common cause of traveler's diarrhea. ETEC diarrhea is most frequently seen in children, suggesting that a protective immune response occurs with age. The pathogenesis of ETEC-induced diarrhea is similar to that of cholera and includes the production of enterotoxins and colonization factors. The clinical symptoms of ETEC infection can range from mild diarrhea to a severe cholera-like syndrome. The effective treatment of ETEC diarrhea by rehydration is similar to treatment for cholera, but antibiotics are not used routinely for treatment except in traveler's diarrhea. The frequency and characterization of ETEC on a worldwide scale are inadequate because of the difficulty in recognizing the organisms; no simple diagnostic tests are presently available. Protection strategies, as for other enteric infections, include improvements in hygiene and development of effective vaccines. Increases in antimicrobial resistance will dictate the drugs used for the treatment of traveler's diarrhea. Efforts need to be made to improve our understanding of the worldwide importance of ETEC.

https://cmr.asm.org/content/cmr/18/3/465.full.pdf

Enterotoxigenic escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention

Properties of bacteriophage PM2: A lipid-containing bacterial virus

A 2-year etiological survey of acute diarrhoea in children aged 0-35 months who were attending treatment facilities was carried out using a standardized protocol in five hospitals in China, India, Mexico, Myanmar, and Pakistan. A total of 3640 cases of diarrhoea and 3279 age- and sex-matched controls were studied; about 60% of the patients were aged less than 1 year and 60% were male. An enteric pathogen was detected in 68% of the cases and in 30% of the controls. In all the study centres, the pathogens most strongly associated with disease were rotavirus (16% of cases, 2% of controls), Shigella spp. (11% of cases, 1% of controls) and enterotoxigenic Escherichia coli (16% of cases, 5% of controls). Rotavirus was commonest among 6-11-month-olds, accounting for 20% of all cases in this age group; 71% of all rotavirus episodes occurred during the first year of life. Shigella spp. were commonest among those aged 12-23 months and 24-35 months, accounting for 22% and 27% of the cases, respectively. The proportion of cases that yielded no pathogen was inversely related to age, being highest (41%) among infants below 6 months of age and lowest (19%) among those aged 24-35 months. These results suggest that microbe-specific intervention strategies for the control of childhood diarrhoeal diseases in developing countries should focus on rotavirus, Shigella spp. and enterotoxigenic E. coli.

Etiology of acute diarrhoea among children in developing countries: a multicentre study in five countries.

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. There is a growing public health concern due to the emergence of a pandemic strain causing severe outbreaks worldwide. Many questions remain unanswered regarding the evolution and population structure of V. parahaemolyticus. In this work, we describe a multilocus sequence typing (MLST) scheme for V. parahaemolyticus based on the internal fragment sequences of seven housekeeping genes. This MLST scheme was applied to 100 V. parahaemolyticus strains isolated from geographically diverse clinical (n = 37) and environmental (n = 63) sources. The sequences obtained from this work were deposited and are available in a public database (http://pubmlst.org/vparahaemolyticus). Sixty-two unique sequence types were identified, and most (50) were represented by a single isolate, suggesting a high level of genetic diversity. Three major clonal complexes were identified by eBURST analysis. Separate clonal complexes were observed for V. parahaemolyticus isolates originating from the Pacific and Gulf coasts of the United States, while a third clonal complex consisted of strains belonging to the pandemic clonal complex with worldwide distribution. The data reported in this study indicate that V. parahaemolyticus is genetically diverse with a semiclonal population structure and an epidemic structure similar to that of Vibrio cholerae. Genetic diversity in V. parahaemolyticus appears to be driven primarily by frequent recombination rather than mutation, with recombination ratios estimated at 2.5:1 and 8.8:1 by allele and site, respectively. Application of this MLST scheme to more V. parahaemolyticus strains and by different laboratories will facilitate production of a global picture of the epidemiology and evolution of this pathogen.

Determination of molecular phylogenetics of Vibrio parahaemolyticus strains by multilocus sequence typing

Analysis of purified simian rotavirus has shown that it contains fewer structural polypeptide classes than previously reported. Two polypeptides (molecular weights, 62,000 and 28,000) commonly found in purified rotaviruses were, in fact, produced by cleavage of a larger structural polypeptide (molecular weight, about 88,000) by trypsin, which is usually employed to increase the yield of rotaviruses in tissue culture. Trypsin-uncleaved, double-shelled rotaviruses are probably composed of only five polypeptide classes; three in the inner layer, and two in the outer layer.

Structural polypeptides of simian rotavirus SA11 and the effect of trypsin.

The DNA molecules isolated from mature bacteriophage PM2 are closed double-stranded rings of a molecular weight of six million Direct evidence for the circularity and supercoiling of PM2 DNA was obtained by electron microscopy Other properties, such as sedimentation behavior at neutral and alkaline pH, unusually high buoyant density in alkaline CsCl, and stability upon heating, are consistent with this structure Only the four common nucleotides are found after hydrolysis and chromatographic analysis of PM2 DNA

https://www.pnas.org/content/pnas/63/4/1164.full.pdf

Romilio T. Espejo

Papers

Properties of bacteriophage PM2: A lipid-containing bacterial virus

Etiology of acute diarrhoea among children in developing countries: a multicentre study in five countries.

Determination of molecular phylogenetics of Vibrio parahaemolyticus strains by multilocus sequence typing

Structural polypeptides of simian rotavirus SA11 and the effect of trypsin.

DNA of bacteriophage PM2: a closed circular double-stranded molecule.