text:
Somerset - Geological Sculpture
The photo shows views of the recently installed wooden sculpture at Staple Hill on the northern edge of the Blackdown Hills in Somerset. Happily the stratigraphy is simple with mainly horizontal bedding and none of the Variscan complexity that we find in west Somerset. One problem was to decide how far the fine detail of the stratigraphy should be shown: do we divide up the Upper Greensand Formation into Foxmould and Chert Beds, or more modern equivalents; was it necessary to show various divisions of the Lower Lias clays as we see them in the cliffs at Charmouth?
A second problem was nomenclature: the British Geological Survey (BGS) Lexicon of Named Rock Units provides BGS definitions of terms that appear on their maps and publications; it revised wholesale the names of formations and members. It was an attempt to bring order into the haphazard terminology that had accumulated over the last 200 years. All very commendable except that well-used names were dumped (e.g. The Yeovil Sands Formation for shame). The Somerset Geology Group is finding problems in advising on which old or new name to use for building stones for the revised Pevsner architectural guide to Somerset. Fortunately, the term 'Upper Greensand Formation' and 'Blue Lias' remain and are widely understood by the public. Ought we to have used the term ´Charmouth Mudstone Formation´ instead of 'Lower Lias clays'? Do we include the term 'formation'?
We decided that traditional semi-descriptive terms were most suitable especially as they include the words 'sand' 'clay´, and 'red marl'. After all we are trying to communicate with the interested public and not frighten them.
The upper photo shows the classic topography: plateau, convex upper slope, linear middle slope, concave lower slope and the vale. For technical reasons it was not possible to show the slip plane of the landslide. The topography is clearly related to the underlying geology: the Greensand makes a resistant capping to the escarpment thus preserving the eye-catching Blackdown Plateau and the Tertiary Clay-with flints and pebble beds that rest on that surface. The pebble beds are shown on the side panel; they are not distinguished on the recently revised BGS Wellington Sheet (2009) and this is a sore point! (Prudden, 2002, 2011 in press, Waters, 1960). They overlie the Clay-with-flints and represent an early Tertiary marine event. They are exposed in ditch sections at Staple Hill. The thicknesses of the pebble beds and the soil have had to be exaggerated on the sculpture. The Lower Lias clays crop out below the Greensand and underlie the clay vale seen in the distance.
The lower photo shows the face of the block. A symbolic landslip is shown. Water percolating through the Greensand emerges where the Greensand rests on impermeable clays; a spring line is shown by a metal strip. These landslides are some of the most extensive in Britain and ring the Blackdown Hills. They are not active today unless loaded or undercut as when the M5 was built; they must date to an environment in the past when melting permafrost resulted in instability. Freborough et al., (2005) point out that many of these landslides tend to have a linear rather than crescent-shaped backscar. Marshy grassland and forest are shown below the spring line.
The sculpture was assembled on the spot from components made in a workshop. The timber was sourced from a fallen oak tree from Neroche Forest. It will be allowed to weather naturally. It is hollow underneath - hedgehogs please note. The sculpture was formed from modules created in a workshop and the components bolted together on site. The sculpture was designed and made by Robert Jakes, sculptor and woodcarver.
Neroche Forest is a Landscape Partnership Scheme supported by the Heritage Lottery Fund and a range of local organisations. Led by the Forestry Commission and working closely with the local community, the Scheme is working to protect and celebrate the heritage of the area, and to provide opportunities for quiet enjoyment, education and training in countryside skills. Neroche Forest is part of the Blackdown Area of Outstanding Beauty. The scheme involving a wooden sculpture was developed by the local stakeholders group.
Visitors may also care to explore via public footpaths the area 2.5km to the east below the site of the Norman Castle Neroche [ST 272 159]. There is a splendid backscar and pressure ridges at the toe of the landslide.
The coast between Charmouth and Lyme Regis shows what Staple Hill would have looked like during periglacial conditions when the landslides were active. Visitors to the coast can clearly distinguish the same rock formations as at Staple Hill and admire the seepages at the base of the Greensand and the active landslides and mudflows. One difference is that the landslides at Charmouth are the result of waves undercutting the cliff.
The Somerset Geology Group was pleased to cooperate in the project as one of its aims is to promote an awareness and understanding of the Somerset's outstanding geodiversity.
Labels: Somerset Geology Group
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Natural Environment of England


Choose what font you want when you personalize your design.
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If you want all capitals, please type it in all capitals.
Because everything is custom made, all clothing items are not returnable.
If you�ve ordered a toy, playing cards, or other non-wearable we will refund in full within 30 days of your receipt, provided it�s in resellable condition.
Click on the button "Clothing Specs"
to check the size before you order.
We're not responsible if we've made a typo in the design dimensions or something like that.
We do all this website stuff ourselves, and do the best we can, but sometimes type in our sleep.
If you've got dogs or kids you probably know what we mean.
We custom make every item for you after you order it. Things are not "off the shelf."
Usual time to ship is about 2 weeks, but please allow up to 6 weeks for delivery.
The last day to order items for Christmas delivery is December 10th.
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We can, many times, alter the coloring of the dog in the design to more closely match that of your dog. If you would like to see if we can do that, email us with the design number and say what you'd like.


Toothpaste On Acne - Does It Help?
Toothpaste On Acne – Does It Help?
While researching the best natural pimple remedies, I've come across some strange things. When I heard about putting toothpaste on acne the first time, I didn't know whether it was serious, or whether it was just a joke. But when a dermatologist confirmed it does work, and there is some science behind it, I decided to investigate a little closer.
For me personally this treatment never worked, but I do know from reading countless of stories online, that plenty of people see good results from this method. So go ahead and try it if you want, but please be aware that it won't for everyone and that results vary depending on your skin type and what kind of spots you have. I had severe acne, which required a lot more than some Colgate!
The reason it works for many people with less severe skin outbreaks is that it dries out the skin and allows the blackheads to heal faster.
Is toothpaste good for acne?
While some people see good results when it comes to reducing the visibility of a zit; there is the question of whether it can actually make things worse if you have a bad skin breakout.
So let's first look at why it works. The ingredients that help with spots are triclosen, baking soda andhydrogen peroxide – all common ingredients in toothpaste. Make sure you use an all-white toothpaste though. The colored versions may not have these active ingredients and may end up causing skin irritation.
A word of warning before using this method. Apply it on a small area first and see how your skin reacts. This will limit the side-effect in case of a bad reaction. Do not use teeth-whitening toothpaste either, as the bleaching agents might make things worse when it comes to irritating sensitive skin. To make things more complex, try finding a paste with little or no fluoride in case of allergic reaction with your skin. If you can afford it, you might want to find a natural, organic, no additives toothpaste to use on your skin to avoid common irritations.
This method is incredibly easy, just quirt some out of the tube and apply to the affected areas. Like I said before, I do recommend using a very small amount on one zit to see what the effects are. It would be risky putting on a lot all over your face the first time.
When trying out the toothpaste acne treatment, always cleanse or wash your face thoroughly and then dry thoroughly. You can leave the toothpaste on for several hours, or for the best results, leave it on overnight.
The benefit of this cure is that even if it doesn't work, you'll smell minty 🙂 Seriously though, try it out, it's free and easy with very little potential side-effects. But stay away from it if you know your skin is very sensitive as it might dry it out too much.


text:
import { Component, DebugElement } from '@angular/core';
import { async, ComponentFixture, TestBed } from '@angular/core/testing';

import { StaticForOf } from '../../../src/utils/staticfor';

@Component({
  selector: 'test-cmp',
  template: `
    <span *staticFor="let item of items">{{item}}</span>
  `
})
// @ts-ignore
class TestComponent {
  items: Iterable<any> = [1, 2];
}

function createTestComponent(): ComponentFixture<TestComponent> {
  return TestBed.createComponent(TestComponent);
}

function getChildrenElements(debugEl: DebugElement) {
  return debugEl.children.map(c => c.nativeElement);
}

// TODO add test for index in context

describe('SimpleForOf', () => {
  let fixture: ComponentFixture<any>;
  let cmp: TestComponent;

  beforeEach(async(() =>
    TestBed.configureTestingModule({
      declarations: [TestComponent, StaticForOf],
    })
  ))

  beforeEach(() => {
    fixture = createTestComponent();
    cmp = fixture.componentInstance;
  })

  afterEach(() => { fixture = null as any; });

  function updateItems(items: any[]) {
    const prevChildrenElements = getChildrenElements(fixture.debugElement);

    cmp.items = items;

    const newChildrenElements = getChildrenElements(fixture.debugElement);

    return {
      prevChildrenElements,
      newChildrenElements,
    };
  }

  function expectChildren(expectedChildren: string[]) {
    fixture.detectChanges();

    const children = getChildrenElements(fixture.debugElement)
      .map(c => c.textContent);

    expect(children).toEqual(expectedChildren);
  }

  it('should reflect initial elements', async(() => {
    expectChildren(['1', '2']);
  }))

  it('should update elements value (reusing same elements)', async(() => {
    const { prevChildrenElements, newChildrenElements } = updateItems([3, 4]);

    expectChildren(['3', '4']);
    expect(prevChildrenElements).toEqual(newChildrenElements);
  }))

  it('sshould reflect update and add new elements', async(() => {
    const { prevChildrenElements, newChildrenElements } = updateItems([1, 2, 3, 4]);

    expectChildren(['1', '2', '3', '4']);
    expect(prevChildrenElements).toEqual(newChildrenElements.slice(0, 2));
  }))

  it('should reflect update and remove extraneous elements', async(() => {
    const { prevChildrenElements, newChildrenElements } = updateItems([1]);

    expectChildren(['1']);
    expect(prevChildrenElements.slice(0, 1)).toEqual(newChildrenElements);
  }))

  it('should work with any iterable', async(() => {
    cmp.items = 'abc';
    expectChildren(['a', 'b', 'c']);

    cmp.items = new Set([1, 2, 3]);
    expectChildren(['1', '2', '3']);
  }))
})

redpajama_set_name: RedPajamaGithub


The Heritage service of Fougères is your contact for group visits in the city and in the castle.
The Tourist Country of Fougères, incoming agency for groups, proposes you organised days to discover Fougères and its Country.
Find here other proposals of our partners for group visits.


text:
10 (десета) година преди новата ера (пр.н.е.) е обикновена година, започваща във вторник, сряда или четвъртък или високосна година, започваща във вторник или сряда по юлианския календар.

Събития

В Римската империя
 Консули на Римската империя са Африкан Фабий Максим и Юл Антоний.
 По нареждане на император Август в Рим за първи път е докаран обелиск от Египет, който служи като символ на римското завоевание на тази страна и като гномон на колосален слънчев часовник изграден на Марсово поле.
 В Панония римляните основават Аквинкум.

В Юдея
 Завършва мащабният строителен проект на Ирод Велики за преобразяване на незначителното селище "Кулата на Стратон". Построеният нов внушителен град е официално осветен и кръстен Цезареа в чест на император Август като към името е прибавено Маритима, за да може той да се отличава от другите градове носещи такова име.

Родени
 1 август – Клавдий, римски император (умрял 54 г.)
 Ирод Агрипа I, цар на Юдея (умрял 44 г.)
 Домиция Лепида, дъщеря на Луций Домиций Ахенобарб и Антония Старата

Починали

Източници

redpajama_set_name: RedPajamaWikipedia


Sweeten up your Christmas digital scrapbook layouts or projects "Sweet Christmas," a collaboration between Snickerdoodle Designs and Linda Cumberland Designs. There's a Santa bringing gifts to everyone, old-fashioned hard candies, and chocolates for all. A whimsical elf looks on as candy canes are hung on snow covered branches. Natural bark, cinnamon, and pinecones round out the delightful Christmas Kit.
Each product is available individually but is also included in the Sweet Christmas Collection, which is your Best Value.


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While none of these organizations "possesses" the Internet collectively these companies determine how it works, and established rules and standards that everyone remains. Contracts and legal framework that underlies all that's happening to ascertain how things work and what happens if something goes wrong. To get a domain name, for example, one needs consent from a Registrar, which has a contract with ICANN. To connect to the Internet, your ISP must be physical contracts with providers of Internet backbone services, and suppliers have contracts with IXPs from the Internet backbone to attach to and with her. Concern over security dilemmas? A working group is formed to focus on the problem and the solution developed and deployed is in the interest of most parties. If the Internet is down, you might have someone to call to get it repaired. If the difficulty is from your ISP, they in turn have contracts in position and service level agreements, which govern the way in which these problems are resolved.
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text:
Madrid and Santander Join Hands for Tourism Promotion
Two Spanish cities; Madrid and Santander join forces for a new journey together in terms of tourism promotion.
The mayor of the capital, José Luis Martínez Almeida, and the mayor of the Cantabrian town, Gema Igual, signed a collaboration agreement to boost the flow of visitors between the two cities and continue developing what is one of its main engines economic and social and cultural development.
As part of the agreement, the two Spanish cities will exchange experiences and knowledge in one of the key economic sectors for the best future development of tourism in Spain.
"It is a protocol that allows us to strengthen relations" said Almeida, who also highlighted that it will enable the city of Madrid to learn from the experience of Santander, one of the cities that has advanced the most in the concept of smart cities, and that it is better taking advantage of the opportunities of innovation and digital transformation. In this sense, the mayor of Madrid has also opted for 'intelligent tourism', as the Santander councilor has described it, and its ability to "monitor tourists and get them to make the decision not only to come, but, also, to return because they have found in these cities the necessary leisure and cultural alternatives".
Work and mutual advice
Since the signing of this protocol, Almeida has valued the constitution of "a work table" led by those responsible for the tourist institutions of both cities for joint work and mutual advice. And, he has highlighted "the exchange of information, knowledge and experience between businessmen from Santander and Madrid" to be in permanent contact and take advantage of synergies through actions such as the launch of specialized forums and reverse promotion days.
On the other hand, the protocol signed also contemplates the establishment of joint projects to give visibility to the two cities, both locally, nationally and internationally.
Madrid and Santander have an attractive offer of gastronomy, leisure and complementary culture that attracts visitors throughout the year, and the actions derived from this agreement will make it possible to promote the dissemination of all these resources in favor of their tourist activity.
With the signing, the city of Madrid, through the Department of Tourism of the City Council, continues to increase its network of alliances to encourage national tourism, which currently represents a little more than half of the total number of travelers that the city receives. city according to data from the first half of the year. In addition to Santander, the capital signed an agreement a few months ago on tourism promotion with Malaga, It also collaborates with the Group of World Heritage Cities of Spain (GCPHE) and plans to continue increasing its alliances in this regard with other Spanish cities.
Tags: Malaga, Madrid, Santander

redpajama_set_name: RedPajamaCommonCrawl


text:
Home » Green Plains acquires two ethanol facilities
Green Plains acquires two ethanol facilities
OMAHA, NEBRASKA, U.S. — Green Plains Renewable Energy, Inc. announced on Nov. 5 that it has entered into a definitive agreement to acquire two ethanol plants of BioFuel Energy Corp. from an entity composed of its lender group.
The ethanol plants are located in Wood River, Nebraska, U.S., and Fairmont, Minnesota, U.S. BioFuel Energy had previously agreed to transfer the ethanol plants to the lender group entity pursuant to a deed in lieu of foreclosure.
Following the transfers, Green Plains will purchase the ethanol plants and certain related assets from the lender group entity. The two facilities have a combined annual production capacity of approximately 220 million gallons. The acquisition will increase Green Plains' production capacity by 28% to over 1 billion gallons of ethanol, 2.9 million tonnes of distillers grains and 230 million pounds of corn oil per year.
Green Plains will purchase the ethanol plants for approximately $101 million, plus working capital at closing. Green Plains intends to fund the purchase with approximately $77 million in term debt and the balance in cash. The closing of the transaction, which is expected to occur during the fourth quarter of 2013, is subject to customary closing conditions and regulatory approvals.
Upon successful completion of the transaction, Green Plains will work to restart the Fairmont plant, which the company anticipates being back in production by the end of 2013. Carl Marks Advisory Group LLC advised the lender group entity in this transaction.
Green Plains to acquire Nebraska ethanol plant
Green Plains acquires grain elevator in northwest Missouri
Green Plains to acquire JW Grain in Nebraska


text:
Boltz4Life
Top News & Blogs
Kendall Jenner Has a "Hannah Montana Room"
August 3, 2020 Boltz4Life	0 Comments
Last week, the masses were permitted a sneak peek into how the supermodel half lives.
First, Gigi Hadid gave us all a glimpse into the chaotic Pinterest board of whimsy that is her personal home aesthetic, sharing a series of images of her eccentrically redecorated apartment on Instagram.
Then, Kendall Jenner showed off her sprawling Los Angeles mansion in a video for Architectural Digest, conducting a tour which included a look at her enigmatically named "Hannah Montana room."
No, the model doesn't have a space exclusively dedicated to Miley Cyrus's alter ego. Just a casual converted bedroom she's dedicated to her daily transformation into an extremely famous person where she also stores a small percentage of her millions of dollars worth of apparel, handbags, and accessories. "So down here, technically, these are two bedrooms, but I needed the space. So I kind of made these into like a fitting room, which I also kind of call my Hannah Montana room and my glam room," Jenner explains.
She also confessed that the Montana boudoir is, "always kind of a mess. This is where I do all my fittings and kind of throw clothes in here. This is where I pack for long trips or trips at all. Even if it is a little bit cluttered in here or a little bit of a mess, I know where everything is."
On top of this Disney Channel-inspired fitting room, Jenner also has a separate huge walk-in closet in her master bedroom that holds her permanent collection of rainbow-colored Hermès Kelly bags and Louboutins. "I don't think I've ever had a closet this big, so I was really stoked about it," she said. "Fun fact, I purge my closet at least every two months. I try and go through it, clean it all out, and it is one of my favorite things to do ever. I feel like a newborn baby every time I do it.
A very fun fact indeed for anyone who happens to know where the closest Goodwill to Kendall's home is located.
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DRONE is the first issue to be published from the Unmanned, Architecture and Security Series research project and publication series.
Drones are unmanned vehicles [UAV]. They are either remotely controlled or, increasingly, autonomously following a pre-programmed mission. Initially, they were developed for use in conflict situations, but the technology also lends itself to a variety of civic purposes, from urban surveillance to monitoring agricultural fields and poaching. UAVs can transport objects, from bombs to books and pizza boxes. In conflict situations they can be used for targeting and killing individuals, but also for providing medical assistance. Drones are cheap to produce and have become faster and smaller in recent years. They can fly solo missions or operate in formation.
This issue presents an overview of the drone technology and the expansion of its operational area, which is strongly connected to the technological evolution triggered by the use of virtual technology and the use of data. Increasingly, decisions are not based on conclusive evidence, but on speculative scenarios.
Contributors: Dan Gettinger, Catherine Harwood, Arthur Holland Mitchel, Yael Messer, Ruben Pater, Francesca Recchia, Gilad Reich, Matthew Stadler, Jane Szita.
This book has been possible with the support of the Graham Foundation for Advanced Studies in the Fine Arts, the Het Nieuwe Instituut, and the Creative Industries Fund NL.


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In 2017, Little Rock City PUMA, AR had a population of 186k people with a median age of N/A and a median household income of $47,482. Between 2016 and 2017 the population of Little Rock City PUMA, AR declined from 186,668 to 186,264, a -0.216% decrease and its median household income grew from $45,510 to $47,482, a 4.33% increase.
The population of Little Rock City PUMA, AR is 45.9% White Alone, 42.4% Black or African American Alone, and 6.83% Hispanic or Latino. 9.98% of the people in Little Rock City PUMA, AR speak a non-English language, and 95% are U.S. citizens.
The largest universities in Little Rock City PUMA, AR are University of Arkansas at Little Rock (2,424 degrees awarded in 2016), University of Arkansas for Medical Sciences (985 degrees), and Baptist Health College-Little Rock (360 degrees).
The median property value in Little Rock City PUMA, AR is $155,000, and the homeownership rate is 56.2%. Most people in Little Rock City PUMA, AR commute by N/A, and the average commute time is 25.1 minutes. The average car ownership in Little Rock City PUMA, AR is N/A per household.
Little Rock City PUMA, AR borders North Little Rock, Sherwood & Jacksonville Cities PUMA, AR and Saline County PUMA, AR.
The economy of Little Rock City PUMA, AR employs 86.9k people. The largest industries in Little Rock City PUMA, AR are Hospitals (9,288 people), Restaurants & Food Services (5,665 people), and Elementary & secondary schools (4,893 people), and the highest paying industries are Offices of physicians ($171,271), Drugs, sundries, & chemical & allied products merchant wholesalers ($168,700), and Electric power generation, transmission & distribution ($135,449).
Median household income in Little Rock City PUMA, AR is $47,482. Males in Little Rock City PUMA, AR have an average income that is 1.48 times higher than the average income of females, which is $47,585. The income inequality in Little Rock City PUMA, AR (measured using the Gini index) is 0.508, which is higher than than the national average.
Households in Little Rock City PUMA, AR have a median annual income of $47,482, which is less than the median annual income of $60,336 across the entire United States. This is in comparison to a median income of $45,510 in 2016, which represents a 4.33% annual growth.
Look at the chart to see how the median household income in Little Rock City PUMA, AR compares to that of it's neighboring and parent geographies.
In 2017, full-time male employees in Little Rock City PUMA, AR made 1.48 times more than female employees.
This chart shows the gender-based wage disparity in the 5 most common occupations in Little Rock City PUMA, AR by number of full-time employees.
In 2017 the highest paid race/ethnicity of Little Rock City PUMA, AR workers was Asian. These workers were paid 1.45 times more than White workers, who made the second highest salary of any race/ethnicity.
This chart shows the race- and ethnicity-based wage disparities in the 5 most common occupations in Little Rock City PUMA, AR by number of full-time employees.
In 2017, the income inequality in Little Rock City PUMA, AR was 0.508 according to the GINI calculation of the wage distribution. Income inequality had a 0.732% growth from 2016 to 2017, which means that wage distribution grew somewhat less even.
The 2017 the GINI for Little Rock City PUMA, AR was higher than than the national average of 0.479. In other words, wages are distributed less evenly in Little Rock City PUMA, AR in comparison to the national average.
This chart shows the number of workers in Little Rock City PUMA, AR across various wage buckets compared to the national average.
In 2016, the PUMA with the highest Median Household Income (Total) in Little Rock City PUMA, AR was N/A with a value of N/A, followed by N/A and N/A, with respective values of N/A and N/A.
The following map shows all of the PUMAs in Little Rock City PUMA, AR colored by their Median Household Income (Total).
N/A% of the population for whom poverty status is determined in Little Rock City PUMA, AR (N/A out of N/A people) live below the poverty line, a number that is approximately the same as the national average of 13.4%. The largest demographic living in poverty are N/A N/A, followed by N/A N/A and then N/A N/A.
The most common racial or ethnic group living below the poverty line in Little Rock City PUMA, AR is false, followed by false and false.
From 2016 to 2017, employment in Little Rock City PUMA, AR grew at a rate of 1.2%, from 85.8k employees to 86.9k employees.
The most common job groups, by number of people living in Little Rock City PUMA, AR, are Miscellaneous managers (2,560 people), Firstline supervisors of retail sales workers (2,537 people), and Registered nurses (2,394 people). This chart illustrates the share breakdown of the primary jobs held by residents of Little Rock City PUMA, AR.
The most common jobs held by residents of Little Rock City PUMA, AR, by number of employees, are Miscellaneous managers (2,560 people), Firstline supervisors of retail sales workers (2,537 people), and Registered nurses (2,394 people).
Compared to other pumas, Little Rock City PUMA, AR has an unusually high number of residents working as Roofers (5.36 times higher than expected), Library technicians (4.44 times), and Appraisers & assessors of real estate (4.02 times).
The highest paid jobs held by residents of Little Rock City PUMA, AR, by average salary, are Physicians & surgeons ($237,527), Chief executives & legislators ($194,324), and Dentists ($169,116).
The most common employment sectors for those who live in Little Rock City PUMA, AR, are Hospitals (9,288 people), Restaurants & Food Services (5,665 people), and Elementary & secondary schools (4,893 people). This chart shows the share breakdown of the primary industries for residents of Little Rock City PUMA, AR, though some of these residents may live in Little Rock City PUMA, AR and work somewhere else. Census data is tagged to a residential address, not a work address.
The most common industries in Little Rock City PUMA, AR, by number of employees, are Hospitals (9,288 people), Restaurants & Food Services (5,665 people), and Elementary & secondary schools (4,893 people).
Compared to other pumas, Little Rock City PUMA, AR has an unusually high number of Data processing, hosting & related services (4.04 times higher than expected), Newspaper publishers (3.77 times), and Administration of human resource programs (3.04 times) industries.
The highest paying industries in Little Rock City PUMA, AR, by average salary, are Offices of physicians ($171,271), Drugs, sundries, & chemical & allied products merchant wholesalers ($168,700), and Electric power generation, transmission & distribution ($135,449).
N/A% of the population of Little Rock City PUMA, AR has health coverage, with N/A% on employee plans, N/A% on Medicaid, N/A% on Medicare, N/A% on non-group plans, and N/A% on military or VA plans.
Per capita personal health care spending in the puma of Little Rock City PUMA, AR was $7,408 in 2014. This is a 6.91% increase from the previous year ($6,929).
Primary care physicians in Little Rock City PUMA, AR see 1522 patients per year on average, which represents a 0.588% decrease from the previous year (1531 patients). Compare this to dentists who see 2218 patients per year, and mental health providers who see 488 patients per year.
Between N/A and N/A, the percent of uninsured citizens in Little Rock City PUMA, AR N/A by N/A from N/A% to N/A%.
The following chart shows how the percent of uninsured individuals in Little Rock City PUMA, AR changed over time compared with the percent of individuals enrolled in various types of health insurance.
Little Rock City PUMA, AR is home to a population of 186k people, from which 95% are citizens. As of 2017, N/A% of Little Rock City PUMA, AR residents were born outside of the country.
The ethnic composition of the population of Little Rock City PUMA, AR is composed of 85.6k White Alone residents (45.9%), 79.1k Black or African American Alone residents (42.4%), 12.8k Hispanic or Latino residents (6.83%), 4.69k Asian Alone residents (2.51%), 3.53k Two or More Races residents (1.89%), 550 Some Other Race Alone residents (0.295%), 333 American Indian & Alaska Native Alone residents (0.178%), and 81 Native Hawaiian & Other Pacific Islander Alone residents (0.0434%).
The most common foreign languages spoken in Little Rock City PUMA, AR are Spanish or Spanish Creole (10,427 speakers), Chinese (1,211 speakers), and Hindi (664 speakers).
In N/A, the median age of all people in Little Rock City PUMA, AR was N/A. Native-born citizens, with a median age of N/A, were generally N/A than foreign-born citizens, with a median age of N/A. But people in Little Rock City PUMA, AR are getting N/A. In N/A, the average age of all Little Rock City PUMA, AR residents was N/A.
As of 2017, N/A% of Little Rock City PUMA, AR residents were born outside of the United States, which is approximately the same as the national average of 13.7%. In 2016, the percentage of foreign-born citizens in Little Rock City PUMA, AR was 6.96%, meaning that the rate has been maintaining.
The following chart shows the percentage of foreign-born residents in Little Rock City PUMA, AR compared to that of it's neighboring and parent geographies.
In 2017, the most common birthplace for the foreign-born residents of Little Rock City PUMA, AR was Mexico, the natal country of 4,170 Little Rock City PUMA, AR residents, followed by India with 1,454 and Guatemala with 919.
As of 2017, 95% of Little Rock City PUMA, AR residents were US citizens, which is higher than the national average of 93.1%. In 2016, the percentage of US citizens in Little Rock City PUMA, AR was 95%, meaning that the rate of citizenship has been decreasing.
The following chart shows US citizenship percentages in Little Rock City PUMA, AR compared to that of it's neighboring and parent geographies.
In 2016, there were 1.08 times more White Alone residents (85.6k people) in Little Rock City PUMA, AR than any other race or ethnicity. There were 79.1k Black or African American Alone and 12.8k Hispanic or Latino residents, the second and third most common racial or ethnic groups.
The following bar chart shows the 8 races and ethnicities represented in Little Rock City PUMA, AR as a share of the total population.
9.98% of Little Rock City PUMA, AR citizens are speakers of a non-English language, which is lower than the national average of 21.5%. In 2015, the most common non-English language spoken in Little Rock City PUMA, AR was Spanish or Spanish Creole. 5.99% of the overall population of Little Rock City PUMA, AR are native Spanish or Spanish Creole speakers. 0.695% speak Chinese and 0.381% speak Spanish or Spanish Creole, the next two most common languages.
Little Rock City PUMA, AR has a large population of military personnel who served in false, N/A times greater than any other conflict.
In 2016, universities in Little Rock City PUMA, AR awarded 4,715 degrees. The student population of Little Rock City PUMA, AR is skewed towards women, with 6,737 male students and 10,891 female students.
Most students graduating from Universities in Little Rock City PUMA, AR are White (2,737 and 60.3%), followed by Black or African American (1,291 and 28.5%), Two or More Races (161 and 3.55%), and Hispanic or Latino (144 and 3.17%).
The largest universities in Little Rock City PUMA, AR by number of degrees awarded are University of Arkansas at Little Rock (2,424 and 51.4%), University of Arkansas for Medical Sciences (985 and 20.9%), and Baptist Health College-Little Rock (360 and 7.64%).
The most popular majors in Little Rock City PUMA, AR are Registered Nursing (676 and 14.3%), Medical Assistant (258 and 5.47%), and General Business Administration & Management (243 and 5.15%).
The median tuition costs in Little Rock City PUMA, AR are $8,760 for private four year colleges, and $6,300 and $14,316 respectively, for public four year colleges for in-state students and out-of-state students.
In 2015, the most common concentation for Bachelors Degree recipients in Little Rock City PUMA, AR was General Business Administration & Management with 166 degrees awarded.
This visualization illustrates the percentage of students graduating with a Bachelors Degree from schools in Little Rock City PUMA, AR according to their major.
In 2016, the Little Rock City PUMA, AR institution with the largest number of graduating students was University of Arkansas at Little Rock with 2,424 degrees awarded.
In 2016, 1,433 men were awarded degrees from institutions in Little Rock City PUMA, AR, which is 0.437 times less than the 3,282 female students who received degrees in the same year.
This chart displays the gender disparity between the top 5 institutions in Little Rock City PUMA, AR by degrees awarded.
In 2016 the majority of degrees awarded at institutions in Little Rock City PUMA, AR were to White students. These 2,737 degrees mean that there were 2.12 times more White students then the next closest race/ethnicity group, Black or African American, with 1,291 degrees awarded.
The median property value in Little Rock City PUMA, AR was $155,000 in 2017, which is 0.712 times smaller than the national average of $217,600. Between 2016 and 2017 the median property value increased from $149,600 to $155,000, a 3.61% increase. The homeownership rate in Little Rock City PUMA, AR is 56.2%, which is lower than the national average of 63.9%. People in Little Rock City PUMA, AR have an average commute time of 25.1 minutes, and they commute by N/A. Car ownership in Little Rock City PUMA, AR is approximately the same as the national average, with an average of N/A per household.
In 2017, the median household income of the N/A households in Little Rock City PUMA, AR grew to $47,482 from the previous year's value of $45,510.
The following chart displays the households in Little Rock City PUMA, AR distributed between a series of income buckets compared to the national averages for each bucket. The largest share of households have an income in the N/A range.
In 2017, the median property value in Little Rock City PUMA, AR grew to to $155,000 from the previous year's value of $149,600.
The following charts display, first, the property values in Little Rock City PUMA, AR compared to it's parent and neighbor geographies and, second, owner-occupied housing units distributed between a series of property value buckets compared to the national averages for each bucket. In Little Rock City PUMA, AR the largest share of households have a property value in the N/A range.
This chart shows the households in Little Rock City PUMA, AR distributed between a series of property tax buckets compared to the national averages for each bucket. In Little Rock City PUMA, AR the largest share of households pay taxes in the < $800 range.
In 2017, 56.2% of the housing units in Little Rock City PUMA, AR were occupied by their owner. This percentage grew from the previous year's rate of 56.2%.
This percentage of owner-occupation is lower than the national average of 63.9%. This chart shows the ownership percentage in Little Rock City PUMA, AR compared it's parent and neighboring geographies.
The following chart displays the households in Little Rock City PUMA, AR distributed between a series of car ownership buckets compared to the national averages for each bucket. The largest share of households in Little Rock City PUMA, AR have N/A, followed by N/A.
Using averages, employees in Little Rock City PUMA, AR have a similar commute time (25.1 minutes) than the normal US worker (25.1 minutes). Additionally, N/A% of the workforce in Little Rock City PUMA, AR have "super commutes" in excess of 90 minutes.
The chart below shows how the median household income in Little Rock City PUMA, AR compares to that of it's neighboring and parent geographies.
In N/A, the most common method of travel for workers in Little Rock City PUMA, AR was N/A, followed by those who N/A and those who N/A.


text:
Characterization, sequencing and comparative genomic analysis of vB_AbaM-IME-AB2, a novel lytic bacteriophage that infects multidrug-resistant Acinetobacter baumannii clinical isolates
Fan Peng1,2,3,
Zhiqiang Mi1,
Yong Huang1,
Xin Yuan2,
Wenkai Niu2,
Yahui Wang1,
Yuhui Hua1,
Huahao Fan1,
Changqing Bai2 &
Yigang Tong1
With the use of broad-spectrum antibiotics, immunosuppressive drugs, and glucocorticoids, multidrug-resistant Acinetobacter baumannii (MDR-AB) has become a major nosocomial pathogen species. The recent renaissance of bacteriophage therapy may provide new treatment strategies for combatting drug-resistant bacterial infections. In this study, we isolated a lytic bacteriophage vB_AbaM-IME-AB2 has a short latent period and a small burst size, which clear its host's suspension quickly, was selected for characterization and a complete genomic comparative study.
The isolated bacteriophage vB_AbaM-IME-AB2 has an icosahedral head and displays morphology resembling Myoviridae family. Gel separation assays showed that the phage particle contains at least nine protein bands with molecular weights ranging 15–100 kDa. vB_AbaM-IME-AB2 could adsorb its host cells in 9 min with an adsorption rate more than 99% and showed a short latent period (20 min) and a small burst size (62 pfu/cell). It could form clear plaques in the double-layer assay and clear its host's suspension in just 4 hours. Whole genome of vB_AbaM-IME-AB2 was sequenced and annotated and the results showed that its genome is a double-stranded DNA molecule consisting of 43,665 nucleotides. The genome has a G + C content of 37.5% and 82 putative coding sequences (CDSs). We compared the characteristics and complete genome sequence of all known Acinetobacter baumannii bacteriophages. There are only three that have been sequenced Acinetobacter baumannii phages AB1, AP22, and phiAC-1, which have a relatively high similarity and own a coverage of 65%, 50%, 8% respectively when compared with our phage vB_AbaM-IME-AB2. A nucleotide alignment of the four Acinetobacter baumannii phages showed that some CDSs are similar, with no significant rearrangements observed. Yet some sections of these strains of phage are nonhomologous.
vB_AbaM-IME-AB2 was a novel and unique A. baumannii bacteriophage. These findings suggest a common ancestry and microbial diversity and evolution. A clear understanding of its characteristics and genes is conducive to the treatment of multidrug-resistant A. baumannii in the future.
Acinetobacter baumanni is a non-fermentative, aerobic, gram-negative bacillus, and is an opportunistic pathogen with global distribution. It is frequently found in elderly patients and cancer patients with compromised immune function, especially in intensive care units. With the use of broad-spectrum antibiotics, immunosuppressive drugs, and glucocorticoids, A. baumannii (AB) has become a major nosocomial pathogen species[1]. Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan drug-resistant (PDR) A. baumannii strains are increasingly prevalent[2]. MDR-AB refers to A. baumannii strains that are resistant to at least three of the following five types of antimicrobial agents: cephalosporins, carbapenems, β-lactamase inhibitors (including piperacillin/tazobactam, cefoperazone/sulbactam, ampicillin/sulbactam), fluoroquinolones, and aminoglycosides[2–4].
Bacteriophage therapy is a potential alternative treatment for multidrug-resistant bacterial infections[5]. A bacteriophage is a bacterial virus that can lyse and kill the host cell. Phage-related studies have gone through three stages. Félix d'Herelle discovered bacteriophage for the treatment of bacterial infections in 1917[6]. After the emergence of antibiotics in the 1940s, phages were seldom used for therapeutic purposes, and mainly functioned as molecular and genetic research tools. With the recent emergence of multidrug-resistant bacteria, however, there has been renewed interest in methods of phage therapy[7]. In this study we isolated a lytic bacteriophage IME-AB2, and compared biological characteristics and genomic sequence with other Acinetobacter baumannii phages. The genomes of A. baumannii phages IME-AB2, A. baumannii AB1, A. baumannii AP22, and A. baumannii phiAC-1 were compared thoroughly in this study. To our knowledge this is the first report of comparison of the characteristics and complete genome sequence of Acinetobacter baumannii bacteriophages. A clear understanding of its genes is conducive to the treatment of multidrug-resistant A. baumannii in the future.
Isolation of a lytic bacteriophage against multidrug-resistant A. baumannii
A. baumannii strain MDR-AB2, isolated from a sputum sample of a patient with pneumonia at PLA Hospital 307, was resistant to multiple antibiotics (Table 1). The bacteria was used to screen bacteriophages in sewage samples from PLA Hospital 307. The isolated phage was designated as vB_AbaM-IME-AB2 following the recommendation by International Committee on Taxonomy of Viruses in phage nomenclature[8]. The pahge IME-AB2 could form clear plaques in the double-layer assay and clear its host's suspension in just 4 hours (Figure 1), indicating that it is a lytic phage. In order to check the development of resistance, we had extended the period of the experiment to 24 h. The result indicated that the bacterial suspension became turbid finally. The final suspension was plated on solid LB culture and then some single bacterial clones were picked to be used for 16 s rDNA sequencing. The sequences of 16 s rDNA proved that the final suspension was A. baumannii that developed resistance to IME-AB2. The phage particles were concentrated with PEG6000 and then purified with a cesium chloride gradients density to a titer of 1 × 1011 pfu/ml. Observation under an electron microscope showed that the phage IME-AB2 consisted of an icosahedral head and a contractile tail. The total length of the phage from the top of the head to the bottom of the tail was about 160 nm, with the head measuring approximately 61.2 nm, and the tail about 90 nm. This morphology suggested that phage IME-AB2 should be classified as a member of the Myoviridae family (Figure. 2). Among the 22 clinical strains of A. baumannii, only three strains of A. baumannii (MDR-AB1139, MDR-AB2 and MDR-AB11) could be lysed by the phage IME-AB2.
Table 1 Antibiotic resistance profile of A. baumannii strain MDR-AB2
The MDR-AB2 suspension at the different optical density (OD600nm) reached to 0.4 from 1.6 and reached to 0.08 from 0.6 respectively after added 200ul IME-AB2 (1 × 10 11 pfu/ml) to the 10 ml MDR-AB2 suspension. It clear its host's suspension in just 4 hours. The control shows increasing OD600nm. The MDR-AB2 suspension added with IME-AB2 finally became turbid in 24 hours.
Transmission electron microscopy of phage IME-AB2. The bidirectional arrows indicated the length of intact phage, phage tail and head. The bar represents a length of 200 nm.
Growth and lytic characteristics of IME-AB2
To determine the optimal multiplicity of infection (MOI) of IME-AB2, the phage and its host cells were mixed at various ratios, and incubated for 3.5 h at 37°C. The results indicated that a MOI of 20 gave the highest production of phage progeny (3.5 × 1011 pfu/ml). To examine the host adsorption ability of phage IME-AB2, host bacteria were infected with IME-AB2 at a MOI of 0.1 and incubated at 37°C. Aliquots were taken at 0, 3, 6, 9, 12, 15, and 18 min post-infection and assayed for the absorbed phage by titration using the double-layer method. The percentages of phage absorption at different time points were plotted (Figure 3a). The results showed that phage IME-AB2 had an adsorption rate of 50% within 3 min, 80% within 6 min and 99% within 9 min.
Biological characteristics of phage IME-AB2. a. Host adsorption ability of phage IME-AB2. b. One-step growth curve of phage IME-AB2.
For one-step growth curve analysis, MDR-AB2 cells (OD600 = 0.3) were infected with phage IME-AB2 at a MOI of 0.1. The bacteriophage was allowed to adsorb for 15 min at 37°C[9]. The mixture was then centrifuged at 12,000 × g for 30 s to remove unadsorbed phage particles, and the resultant pellet was re-suspended in 5 ml of LB medium. Samples were incubated at 37°C and collected every 10 min during 0–60 min, as well as at 90 and 120 min[10]. As shown in Figure 3b, the latent period of phage IME-AB2 lasted for 20 min, the burst period reached a peak at 30 min, and the phage multiplication reached the final plateau phase at 50 min. The burst size of phage IME-AB2 was determined to be 62 pfu/cell (burst size = the total number of phages liberated at the end of one cycle of growth /the number of infected bacteria)[11].
Analysis of the phage proteins and genome
Purified phage particles were denatured in loading buffer (50 mM Tris–HCl, 2% Sodium Dodecyl Sulfate-polyacrylamide, 0.1%Bromophenol blue, 10% Glycerol and 1% β-Mercaptoethanol) and heated in a boiling water bath for 5 min, followed by separation of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results indicated that the structural proteins of phage IME-AB2 showed a pattern of nine protein bands in 10% SDS-PAGE gel, with molecular masses ranging from 15–100 kDa (Figure 4a). The most abundant protein band in the gel above 35 kDa was analyzed with liquid sampling Mass Spectrometry (LS-MS) and proved to be the phage putative capsid protein.
Protein and genomic DNA analysis of phage IME-AB2. a. SDS-PAGE gel (10%) of whole protein from phage IME-AB2. Molecular weights of protein marker was indicated by lines. b. Endonuclease digestion analysis of phage IME-AB2 genomic DNA. Phage IME-AB2 genomic DNA was digested with the restriction enzyme Nde I, HincII and HindIII. The digested DNA fragments were separated by 1% agarose gel electrophoresis. M, DNA molecular weight marker; Lanes 1, undigested phage IME-AB2 genomic DNA; lane 2, 3, 4, genomic DNA digested with NdeI , HincII and HindIII, respectively.
The genome analysis indicated that phage IME-AB2 has a double-stranded DNA genome, approximately 40 kb in size. The genome of phage IME-AB2 could be digested with endonuclease Nde I, HincII and HindIII (Figure 4b). It was found that endonuclease enzymes, HindIII and HincII, have the 35 and 16 cutting sites on the genome of phage IME-AB2 respectively by Vector NTI[12]. Compared to other A. baumannii complete genome , the two endonucleases also have most restriction enzyme cutting sites on them.
High-throughput sequencing of the phage genomic DNA generated 311,503 valid reads with which the complete sequence of the genome was assembled using both Velvet and CLC Genomic Workbench, with an average coverage of 785 × [13]. The complete genome of phage IME-AB2 consists of 43,665 bp, with an average GC content of 37.5% (Figure 5). Annotation results showed that the genome encodes 82 coding sequences (CDSs) (GenBank Assession number: JX976549). The classification of the 82 CDSs is shown in Table 2 and Figure 6. The complete genome of IME-AB2 is organized into three functional units which encoding structural proteins, metabolic proteins and packaging-associated proteins respectively. No tRNA was found in the genome of IME-AB2, and no significant proteins considered to be markers of temperate bacteriophages were identified. Running blastn showed that the isolated IME-AB2 has a high similarity to Acinetobacter phage AB1 (Genbank Accession Number: HM368260.1), Acinetobacter phage AP22 (Genbank Accession Number: HE806280.1) and Acinetobacter phage phiAC-1 (GenBank accession number: JX560521), which were isolated in China, Russia and Korea respectively. The phage AB1, AP22 or phiAC-1 has a genome of about 45 kb and owns a coverage of 65%, 50%, 8% respectively when compared with the isolated phage IME-AB2. Genomic annotation found that IME-AB2 encodes 82 CDSs, AB1 85 CDSs, AP22 89 CDSs, phiAC-1 82 CDSs. The 82 CDSs from IME-AB2 shared 63 homologues with AB1, 60 homologues with AP22 and 36 homologues with phiAC-1 respectively (Table 3). Totally, 22 of the 82 CDSs encoded by IME-AB2 were identified to be putatively functional. Genomic analysis revealed that the bacteriophage IME-AB2 was most closely related to AB1. A nucleotide alignment of the four Acinetobacte r baumannii phages showed that some functional regions are highly homologous, with no significant rearrangements observed (Table 3 and Figure 7). It revealed a stable area. Stability is suggested from the high level of nucleotide identity, lack of inversions and other major rearrangements, and the stabilizing selection inferred for virtually all genes harboring synonymous and non-synonymous mutations[14]. Functional related genes are sequential, yet there are a lot of breakpoint modules obviously and some sections of these strains of phages are nonhomologous (Table 3 and Figure 7). It illustrated that these structural genes had occurred in the extensive structural rearrangements during evolution. Bacteriophages are the most diverse and abundant biological entities in nature environment. Most of them can hardly to be found homologous to another bacteriophage, which means evolutionary success obtained by bacteriophages. Furthermore, the diversity is such that even genes with required functions cannot always be recognized. During bacteriophage evolution,the elimination or recombination of genes result in the diversity and meanwhile confer a selective advantage to survive and infect so that phages can better adapt to host bacteria.
Circular map of the IME-AB2 genome prepared using CGView. The outer ring denotes the IME-AB2 genome and CDSs. The inner rings show G + C content and G + C skew, where peaks represent the positive (outward) and negative (inward) deviation from the mean G + C content and G + C skew, respectively.
Table 2 Functional classification of the 82 CDSs in the IME-AB2 genome
Genome map of phage IME-AB2. Arrows indicate putative CDSs, along with their orientations. Functionally assigned genes are differently colored (purple, structural gene; green, metabolic gene; orange, replication/packaging-associated gene; blue, other gene). Promoters are illustrated as green arrowheads, while terminators are displayed as red arrowheads.
Table 3 Comparative genomic analysis of A. baumannii phage IME-AB2, A. baumannii phage AB1, A. baumanni i phage AP22, and A3 baumannii phage phiAC-1
Multiple genome alignment performed using Mauve software ( http://asap.ahabs.wisc.edu/mauve/ ) and the chromosomes of A. baumannii IME-AB2, A. baumannii AB1, A. baumannii AP22, and A. baumannii phiAC-1. IME-AB2 is the reference for alignments and comparisons to the three other strains. Boxes with identical colors represent local colinear blocks (LCB), indicating homologous DNA regions shared by two or more chromosomes without sequence rearrangements. LCBs indicated below the horizontal black line represent reverse complements of the reference LCB.
Structural proteins
Seven CDSs encoding structural proteins were identified in the phage IME-AB2. The putative capsid protein (CDS.12) is similar to that of phage AB1 (gp15), phage AP22 (gp30), phage phiAC-1 (0043). Phage AB1 (gp27), phage AP22 (gp18), phage phiAC-1 (0031) also share homology to the putative phage head protein encoded by IME-AB2 CDS.24. CDS.71 and CDS.72 of phage IME-AB2 are identified to be associated with tail fiber protein. CDS.74 and CDS.77 of phage IME-AB2 are predicted to encode proteins responsible for baseplate. These two related proteins can be found similar area in the other three phages. The results also demonstrate the phage tail related proteins generally cluster together (Table 3).
Metabolic proteins
A unique feature of the IME-AB2 genome is that it encodes cobalt transport protein (CDS.6). Notably, cobalt is a cofactor and is required by enzymes from bacteria[15]. It is possible that these metabolic enzymes benefit phage by enhancing the metabolism of the infected bacterial cell, which could in turn increase phage proliferation. No similar cobalt proteins were found in the other three phages sharing homology with IME-AB2. CDS.8 encodes a putative RNA polymerase protein. It is necessary for constructing RNA chains using DNA genes as templates, a process called transcription. Transcription of most double-stranded DNA bacteriophages rely on their host bacteria[16]. The putative CDS.33 of IME-AB2 is predicted to encode HTH domains which have been recruited to a wide range of functions beyond transcription regulation, such as DNA repairing and replication, RNA metabolism and protein-protein interactions in diverse signaling contexts. Beyond their basic role in mediating macromolecular interactions, the HTH domains have also been incorporated into the catalytic domains of diverse enzymes[17]. In functional terms, the HNH endonuclease domain (CDS.52) is found in CRISPR-related proteins. CRISPR functions as a prokaryotic immune system, in that it confers resistance to exogenous genetic elements[18]. CDS.47 is predicted to be a putative bacteriophage-associated immunity protein, which was considered to be responsible for phage superinfection immunity[19]. CDS.68 and CDS.81 are putative lysozyme family protein. Unlike, CDS.0057, 0058, 0059 encoded by phiAC-1, which are putative lysozyme-like domain protein and adjacent to each other, the two lysozyme-like proteins from IME-AB2 are not clustered.
Packaging-associated proteins
In the four similar Acinetobacter baumannii phages, the genetic elements encoding the products involved in the packaging system are commonly found adjacent to one another in the phage genomes. Packaging system is generally composed of big subunit (CDS.27) and small subunit (CDS.28). Usually, the two subunits of the terminase and head portal protein (CDS.26) are closely connected in the packaging system, while the portal protein is a bacteriophage component that forms a hole, or portal, enabling DNA passage during packaging and ejection[20]. It also forms the junction between the phage head (capsid) and the tail proteins.
With the emergence of a growing number of drug-resistant bacterial species, and the difficulties surrounding the development of novel antibiotics[21], exploring novel or alternative therapeutic methods is imperative. The recent renaissance of bacteriophage therapy may provide new treatment strategies for combatting drug-resistant bacterial infections. Although a large number of work on phage therapy in human disease had been done[22–24], the host-specific infection and the relatively narrow lytic spectrum of phage is one of the obstacles to their further application. Individualized phage therapy may represent the future of phage therapy, where bacterial infections will be treated with phage combinations that have already been shown as effective for that particular bacterium. To overcome these limitations, strategies such as screening more lytic phages, combining phages with antibiotics, or administrating phages cocktails should be investigated[25, 26]. Therefore, it is very important to isolate novel and sensitive phages to enrich the phage arsenal[27].
All known Acinetobacter baumannii bacteriophages were summarized and compared in this research. There are nearly 20 A. baumannii phage strains reported in the literature mainly in 2012. Most of the phages genome length are about 40 kb. Just only thirteen complete A. baumannii phage genomes were sequenced and deposited in the GenBank database currently. Five of those genomes consist of an approximately 160 kb linear DNA molecule (Acinetobacter phage Ac42,Acj61,Acj9,133,and ZZ1), and are annotated as T4-like phage[5, 28]. The remaining eight phages contain a genome of 30–50 kb, and may be classified into two different groups according to sequence similarity. In one group, there are four phages with a linear genome, including phiAB1, which was already classified as a ϕKMV-like virus[29], phage YMC/09/02/B1251_ABA_BP[30], Acinetobacter phage AB3 and Acinetobacter phage Abp1. The other group includes four phages: AB1 (GenBank accession number: HM368260), AP22 (GenBank accession number: HE806280), phiAC-1 (GenBank accession number: JX560521), and our phage IME-AB2. We compared the growth characteristics and the genome of A. baumannii phage as shown in Table 4. The results indicated that the newly isolated phage IME-AB2 has a shorter latent and burst period than AB1 and AP22, which implied that the IME-AB2 was more lytic and the burst size produced by IME-AB2 was smaller than those phages[31]. All the listed A. baumannii bacteriophages or its lysin had been tested in treating bacterial infection such as inhibiting biofilm formation or effecting on host cell survival. Although the bacteriophage IME-AB2 could lyse the host bacteria and cleared the bacterial suspension in 4 hours, we observed that the resistant bacteria appeared and made the suspension turbid again in 24 hours. The easily emerging resistant bacteria after infection with phage might be an obstacle when fighting against bacterial infection with bacteriophage in the future.
Table 4 Comparative analysis of all known Acinetobacter baumannii bacteriophages
A lytic A. baumannii bacteriophage IME-AB2 was isolated and characterized in this research. The complete genome of IME-AB2 was sequenced and compared to those of A. baumannii phage AB1, A. baumannii phage AP22, and A. baumannii phage phiAC-1 in detail. The genome of IME-AB2 was replete with novel genes without known relatives, which indicated that IME-AB2 was a novel and unique A. baumannii bacteriophage. Although the resistant A. baumannii appeared finally after infection with IME-AB2, the comprehensive understanding of the phage's characteristics is conducive to the treatment of multidrug-resistant A. baumannii in the future.
Bacterial strains, Phage isolation, propagation, and titration
This study included 22 clinical strains of A. baumannii (MDR-AB1139, MDR-AB1, MDR-AB2, MDR-AB3, MDR-AB4, MDR-AB5, … , MDR-AB19,MDR-AB20, MDR-AB21). All the clinical samples were taken as part of standard patient care at the PLA Hospital 307, Beijing, China. The patients were orally informed that the specimens would be used for screening bacteria and the tests were optional on laboratory sheet. Blood, sputum and skin swabs were collected from patients with consent under the Ethics Committee of the PLA Hospital 307. The protocol of screening bacteria was approved by the Ethics Committee of the PLA Hospital 307 and Beijing Institute of Microbiology and Epidemiology Ethics Committee.
Multidrug-resistant A. baumannii strain MDR-AB2 was used as an indicator for bacteriophage screening of raw sewage samples collected from PLA Hospital 307. Sewage samples were separated by centrifugation at 12,000 × g for 20 min. Following removal of the solid impurities by centrifugation, the supernatants were filtered through a 0.45 μm pore-size membrane filter to remove bacterial debris. Filtrate (4 ml) was added to 2 ml of 3× Luria-Bertani (LB) broth medium and mixed with 0.1 ml of A. baumannii overnight culture (OD600 = 0.6) to enrich the phage at 37°C overnight. Following enrichment, the culture was centrifuged at 12,000 × g for 10 min, and then the supernatant was filtered with a 0.45 μm pore-size membrane filter to remove the residual bacterial cells. The filtrate (0.1 ml) was mixed with 0.5 ml of A. baumannii in LB culture (OD600 = 0.6) and 5 ml of molten top soft nutrient agar (0.75% agar), which was then overlaid onto solidified base nutrient agar (1.5% agar)[43]. Following incubation for 6 h at 37°C, the clear phage plaques were picked from the plate. The phage titer was determined using the double-layered method previously described by Adams[44].
Phage concentration , purification and storage
A single plaque was picked into 5 ml of LB medium containing MDR-AB2 (OD600 = 0.6) and cultured at 37°C for 6 h. A 5 ml aliquot of suspension was transferred into 500 ml of LB medium for culture at 37°C overnight. Chloroform was then added to the 500 ml of culture to a final concentration 0.1% before being mixed gently and allowed to stand at room temperature for about 30 min. Solid NaCl was added to the culture to a final concentration of 1 M, which was then incubated in an ice water bath for 1 h. The culture was centrifuged at 11,000 × g for 10 min to remove cell debris, and polyethylene glycol 6000 (PEG6000) was added to the supernatant to a final concentration of 10% (w/v) while slowly stirring with a magnetic stirrer at room temperature. This solution was transferred to a polypropylene centrifuge tube in an ice water bath and incubated at least 1 h to precipitate the phage particles. Following centrifugation (11,000 × g for 10 min at 4°C), the phage-containing precipitate was resuspended in 5 ml of SM buffer (50 mM Tris-Cl, 100 mM NaCl, 8 mM MgSO4, pH 7.5)[45]. An equal volume of chloroform was then added to separate the phage particles from PEG6000. Following centrifugation at 3,000 × g for 10 min, the aqueous phase was recovered and filtered through a 0.22 μm pore-size membrane filter to remove debris. The concentrated 1.0 ml of phage suspensions were layered on the top of a cesium chloride gradient solutions (density of 1.3 g/ml-0.45 g of cesium chloride in 1.0 ml of water; density of 1.5 g/ml-0.83 g of cesium chloride in 1.0 ml of water; density of 1.7 g/ml-1.28 g of cesium chloride in 1.0 ml of water) in 5.0 ml cellulose nitrate centrifuge tube[46]. After centrifugation in a Beckman Coulter Swinging Bucket Rotor (SW41, Ti) for 40 min at 100,000 × g, the concentrated phages at the visible band were collected by means of a capillary pipette. The purified phage was stored at 4°C.
Determination of lytic spectrum
The host range was determined by spot test. Briefly, 0.5 ml of bacterial overnight culture was mixed with 5 ml of molten top soft nutrient agar (0.75% agar) and then overlaid on the surface of solidified base nutrient agar (1.5% agar). Once the top layer also solidified, 2 μl of the phage preparation(1 × 109 pfu/ml) was spotted onto the plate, which was incubated for 6 h at 37°C.
Phage stock solution was directly stained with phosphotungstic acid (PTA) for 2 min. After being dried at room temperature, the grid was examined using a Philips TECNAI-10 transmission electron microscope (TEM) to observe and record the morphology of the phage particles[41].
Extraction of phage genomic DNA
Purified phage particles were treated with DNase I (1 μg/ml) (Takara) and RNase A (1 μg/ml) (Takara) for 30 min, and then the nucleases were inactivated at 80°C for 15 min. Ethylene diamine tetraacetic acid (EDTA) (20 mM), proteinase K (50 μg/ml) and sodium dodecyl sulfate (SDS) (0.5%) were then added and the mixture was incubated at 56°C for 1 h. Phage lysate was extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). Chloroform extraction was repeated until there was no phenol odor. An equal volume of isopropanol (AR grade) was added and the sample was incubated overnight at -20°C to precipitate the phage genomic DNA. The pellet was washed with 75% ethanol, and then deionized water was used to dissolve the precipitated genomic DNA.
Whole genome sequence and bioinformatics analysis
The genomic DNA of IME-AB2 was subjected to high-throughput sequencing using a Life Technologies Ion Personal Genome Machine Ion Torrent sequencer (San Francisco, CA) according to the manufacturer's instructions. The complete genome sequence of phage IME-AB2 was assembled using Velvet[47] and CLC Bio (Aarhus, Denmark), and annotated using RAST[48] and InterPro[49]. Sequence similarity analysis and comparison were performed using NCBI packages.
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This research was supported by a grant from the National Natural Science Foundation of China (No. 81072350), the National Hi-Tech Research and Development (863) Program of China (No. 2012AA022-003), the China Mega-Project on Major Drug Development (No. 2011ZX09401-023), the China Mega-Project on Infectious Disease Prevention (No. 2013ZX10004-605 and No. 2011ZX10004-001), the State Key Laboratory of Pathogen and BioSecurity Program (No. SKLPBS1113), and the Particular Clinical Applied Research of the Capital of China (No.Z121107001012127).
Nucleotide sequence accession number: The whole-genome sequence of phage vB_AbaM-IME-AB2 has been deposited in the NCBI nucleotide sequence database under GenBank assession number: JX976549.
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, 100071, China
Fan Peng, Zhiqiang Mi, Yong Huang, Yahui Wang, Yuhui Hua, Huahao Fan & Yigang Tong
Department of Respiratory Medicine, PLA Hospital 307, Beijing, 100071, China
Fan Peng, Xin Yuan, Wenkai Niu & Changqing Bai
The Third Xiangya Hospital of Central South University, Changsha, 410013, China
Fan Peng
Zhiqiang Mi
Yong Huang
Xin Yuan
Wenkai Niu
Yahui Wang
Yuhui Hua
Huahao Fan
Changqing Bai
Yigang Tong
Correspondence to Changqing Bai or Yigang Tong.
Fan Peng isolated the phage, identified the characterization, and drafted the manuscript. Zhiqiang Mi and Yong Huang were responsible for sequencing and drafting the manuscript. Wenkai Niu and Xin Yuan collected clinical bacteria. Yahui Wang conducted the phage concentration and purification. Yuhui Hua and Huahao Fan conducted EM assay. Yigang Tong and Changqing Bai conceived and designed the experiments. All authors read and approved the final manuscript.
Fan Peng, Zhiqiang Mi, Yong Huang contributed equally to this work.
Peng, F., Mi, Z., Huang, Y. et al. Characterization, sequencing and comparative genomic analysis of vB_AbaM-IME-AB2, a novel lytic bacteriophage that infects multidrug-resistant Acinetobacter baumannii clinical isolates. BMC Microbiol 14, 181 (2014). https://doi.org/10.1186/1471-2180-14-181
Bacteriophage
Microbial genetics, genomics and proteomics


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