Phylogenetic analysis and revision of subfamily classification of Belostomatidae genera (Insecta: Heteroptera: Nepomorpha), Zoological Journal of the Linnean Society, Oxford Academic

Phylogenetic analysis and revision of subfamily classification of Belostomatidae genera (Insecta: Heteroptera: Nepomorpha)

JosÉ Ricardo I Ribeiro, Shin-Ya Ohba, Dominique Pluot-Sigwalt, Fabiano Stefanello, Wenjun Bu, Solange E Meyin-A-Ebong, Eric Guilbert, Phylogenetic analysis and revision of subfamily classification of Belostomatidae genera (Insecta: Heteroptera: Nepomorpha), Zoological Journal of the Linnean Society, Volume 182, Issue 2, February 2018, Pages 319–359, https://doi.org/10.1093/zoolinnean/zlx041

Abstract

Recent investigations into relationships among the cosmopolitan Belostomatidae have led to recognition of the Belostomatinae, Horvathiniinae and Lethocerinae clades. Here we investigate the relationships among the genera of this family (with Appasus, Benacus and Kirkaldyia now resurrected, and three fossils: Cratonepa, Iberonepa and Lethocerus vetus) by including modifications or clarifications of both somatic and genitalic characters (including some spermatheca traits), as well as multiple genes (COI, 18S rDNA and 16S rDNA). A comparative study of these genera yielded putative homology hypotheses coded as 104 morphological characters and 1829 aligned characters. A majority-rule tree utilizing the Bayesian method [the ‘master’ tree with highlighted clades estimated by maximum parsimony and maximum likelihood (ML)] is as follows: (Cratonepa, Iberonepa, (Benacus, Kirkaldyia, Lethocerus), (((Horvathinia, Hydrocyrius), Limnogeton), (((Abedus, Weberiella), Belostoma), (Appasus, Diplonychus))))). Lethocerinae and Belostomatinae (now with Horvathinia included) form two monophyletic groups. Weberiella appears as the sister group of Abedus, and Belostomatini had to be reformulated. In all trees, the monophyly of Diplonychinitrib. nov. (Appasus+Diplonychus) was recovered with high support indices. This study highlights a global tree based on combining the molecular and morphological data representing topologies from separate analyses, irrespective of the existence of missing data and the type of dataset.

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Ranatra rod-shaped: a description of the appearance and biological development of a water bug

At the beginning of this chapter, we described how porcelain Chamberland filters with pores small enough to allow viruses to pass through were used to discover TMV. Today, porcelain filters have been replaced with membrane filters and other devices used to isolate and identify viruses.

Isolation of Viruses

Unlike bacteria, many of which can be grown on an artificial nutrient medium, viruses require a living host cell for replication. Infected host cells (eukaryotic or prokaryotic) can be cultured and grown, and then the growth medium can be harvested as a source of virus. Virions in the liquid medium can be separated from the host cells by either centrifugation or filtration. Filters can physically remove anything present in the solution that is larger than the virions; the viruses can then be collected in the filtrate (see Figure 1).

Figure 1. Membrane filters can be used to remove cells or viruses from a solution. (a) This scanning electron micrograph shows rod-shaped bacterial cells captured on the surface of a membrane filter. Note differences in the comparative size of the membrane pores and bacteria. Viruses will pass through this filter. (b) The size of the pores in the filter determines what is captured on the surface of the filter (animal [red] and bacteria [blue]) and removed from liquid passing through. Note the viruses (green) pass through the finer filter. (credit a: modification of work by U.S. Department of Energy)

Think about It

  • What size filter pore is needed to collect a virus?

Cultivation of Viruses

Viruses can be grown in vivo (within a whole living organism, plant, or animal) or in vitro (outside a living organism in cells in an artificial environment, such as a test tube, cell culture flask, or agar plate). Bacteriophages can be grown in the presence of a dense layer of bacteria (also called a bacterial lawn) grown in a 0.7 % soft agar in a Petri dish or flat (horizontal) flask (see Figure 2). The agar concentration is decreased from the 1.5% usually used in culturing bacteria. The soft 0.7% agar allows the bacteriophages to easily diffuse through the medium. For lytic bacteriophages, lysing of the bacterial hosts can then be readily observed when a clear zone called a plaque is detected (see Figure 2). As the phage kills the bacteria, many plaques are observed among the cloudy bacterial lawn.

Figure 2. (a) Flasks like this may be used to culture human or animal cells for viral culturing. (b) These plates contain bacteriophage T4 grown on an Escherichia coli lawn. Clear plaques are visible where host bacterial cells have been lysed. Viral titers increase on the plates to the left. (credit a: modification of work by National Institutes of Health; credit b: modification of work by American Society for Microbiology)

Animal viruses require cells within a host animal or tissue-culture cells derived from an animal. Animal virus cultivation is important for 1) identification and diagnosis of pathogenic viruses in clinical specimens, 2) production of vaccines, and 3) basic research studies. In vivo host sources can be a developing embryo in an embryonated bird’s egg (e.g., chicken, turkey) or a whole animal. For example, most of the influenza vaccine manufactured for annual flu vaccination programs is cultured in hens’ eggs.

The embryo or host animal serves as an incubator for viral replication (see Figure 3). Location within the embryo or host animal is important. Many viruses have a tissue tropism, and must therefore be introduced into a specific site for growth. Within an embryo, target sites include the amniotic cavity, the chorioallantoic membrane, or the yolk sac. Viral infection may damage tissue membranes, producing lesions called pox; disrupt embryonic development; or cause the death of the embryo.

Figure 3. (a) The cells within chicken eggs are used to culture different types of viruses. (b) Viruses can be replicated in various locations within the egg, including the chorioallantoic membrane, the amniotic cavity, and the yolk sac. (credit a: modification of work by “Chung Hoang”/YouTube)

For in vitro studies, various types of cells can be used to support the growth of viruses. A primary cell culture is freshly prepared from animal organs or tissues. Cells are extracted from tissues by mechanical scraping or mincing to release cells or by an enzymatic method using trypsin or collagenase to break up tissue and release single cells into suspension. Because of anchorage-dependence requirements, primary cell cultures require a liquid culture medium in a Petri dish or tissue-culture flask so cells have a solid surface such as glass or plastic for attachment and growth. Primary cultures usually have a limited life span. When cells in a primary culture undergo mitosis and a sufficient density of cells is produced, cells come in contact with other cells. When this cell-to-cell-contact occurs, mitosis is triggered to stop. This is called contact inhibition and it prevents the density of the cells from becoming too high. To prevent contact inhibition, cells from the primary cell culture must be transferred to another vessel with fresh growth medium. This is called a secondary cell culture. Periodically, cell density must be reduced by pouring off some cells and adding fresh medium to provide space and nutrients to maintain cell growth. In contrast to primary cell cultures, continuous cell lines, usually derived from transformed cells or tumors, are often able to be subcultured many times or even grown indefinitely (in which case they are called immortal). Continuous cell lines may not exhibit anchorage dependency (they will grow in suspension) and may have lost their contact inhibition. As a result, continuous cell lines can grow in piles or lumps resembling small tumor growths (see Figure 4).

Figure 4. Cells for culture are prepared by separating them from their tissue matrix. (a) Primary cell cultures grow attached to the surface of the culture container. Contact inhibition slows the growth of the cells once they become too dense and begin touching each other. At this point, growth can only be sustained by making a secondary culture. (b) Continuous cell cultures are not affected by contact inhibition. They continue to grow regardless of cell density. (credit “micrographs”: modification of work by Centers for Disease Control and Prevention)

An example of an immortal cell line is the HeLa cell line, which was originally cultivated from tumor cells obtained from Henrietta Lacks, a patient who died of cervical cancer in 1951. HeLa cells were the first continuous tissue-culture cell line and were used to establish tissue culture as an important technology for research in cell biology, virology, and medicine. Prior to the discovery of HeLa cells, scientists were not able to establish tissue cultures with any reliability or stability. More than six decades later, this cell line is still alive and being used for medical research. See “The Immortal Cell Line of Henrietta Lacks” below to read more about this important cell line and the controversial means by which it was obtained.

Think about It

  • What property of cells makes periodic dilutions of primary cell cultures necessary?

The Immortal Cell Line of Henrietta Lacks

In January 1951, Henrietta Lacks, a 30-year-old African American woman from Baltimore, was diagnosed with cervical cancer at John Hopkins Hospital. We now know her cancer was caused by the human papillomavirus (HPV). Cytopathic effects of the virus altered the characteristics of her cells in a process called transformation, which gives the cells the ability to divide continuously. This ability, of course, resulted in a cancerous tumor that eventually killed Mrs. Lacks in October at age 31. Before her death, samples of her cancerous cells were taken without her knowledge or permission. The samples eventually ended up in the possession of Dr. George Gey, a biomedical researcher at Johns Hopkins University. Gey was able to grow some of the cells from Lacks’s sample, creating what is known today as the immortal HeLa cell line. These cells have the ability to live and grow indefinitely and, even today, are still widely used in many areas of research.

Figure 5. A multiphoton fluorescence image of HeLa cells in culture. Various fluorescent stains have been used to show the DNA (cyan), microtubules (green), and Golgi apparatus (orange). (credit: modification of work by National Institutes of Health)

According to Lacks’s husband, neither Henrietta nor the family gave the hospital permission to collect her tissue specimen. Indeed, the family was not aware until 20 years after Lacks’s death that her cells were still alive and actively being used for commercial and research purposes. Yet HeLa cells have been pivotal in numerous research discoveries related to polio, cancer, and AIDS, among other diseases. The cells have also been commercialized, although they have never themselves been patented. Despite this, Henrietta Lacks’s estate has never benefited from the use of the cells, although, in 2013, the Lacks family was given control over the publication of the genetic sequence of her cells.

This case raises several bioethical issues surrounding patients’ informed consent and the right to know. At the time Lacks’s tissues were taken, there were no laws or guidelines about informed consent. Does that mean she was treated fairly at the time? Certainly by today’s standards, the answer would be no. Harvesting tissue or organs from a dying patient without consent is not only considered unethical but illegal, regardless of whether such an act could save other patients’ lives. Is it ethical, then, for scientists to continue to use Lacks’s tissues for research, even though they were obtained illegally by today’s standards?

Ethical or not, Lacks’s cells are widely used today for so many applications that it is impossible to list them all. Is this a case in which the ends justify the means? Would Lacks be pleased to know about her contribution to science and the millions of people who have benefited? Would she want her family to be compensated for the commercial products that have been developed using her cells? Or would she feel violated and exploited by the researchers who took part of her body without her consent? Because she was never asked, we will never know.

Detection of a Virus

Regardless of the method of cultivation, once a virus has been introduced into a whole host organism, embryo, or tissue-culture cell, a sample can be prepared from the infected host, embryo, or cell line for further analysis under a brightfield, electron, or fluorescent microscope. Cytopathic effects (CPEs) are distinct observable cell abnormalities due to viral infection. CPEs can include loss of adherence to the surface of the container, changes in cell shape from flat to round, shrinkage of the nucleus, vacuoles in the cytoplasm, fusion of cytoplasmic membranes and the formation of multinucleated syncytia, inclusion bodies in the nucleus or cytoplasm, and complete cell lysis (see Table 1).

Further pathological changes include viral disruption of the host genome and altering normal cells into transformed cells, which are the types of cells associated with carcinomas and sarcomas. The type or severity of the CPE depends on the type of virus involved. Table 1 lists CPEs for specific viruses.

Table 1. Cytopathic Effects of Specific Viruses [1]
Virus Cytopathic Effect Example
Paramyxovirus Syncytium and faint basophilic cytoplasmic inclusion bodies
Poxyvirus Pink eosinophilic cytoplasmic inclusion bodies (arrows) and cell swelling
Herpesvirus Cytoplasmic stranding (arrows) and nuclear inclusion bodies (dashed arrow)
Adenovirus Cell enlargement, rounding, and distinctive grape-like clusters

Hemagglutination Assay

A serological assay is used to detect the presence of certain types of viruses in patient serum. Serum is the straw-colored liquid fraction of blood plasma from which clotting factors have been removed. Serum can be used in a direct assay called a hemagglutination assay to detect specific types of viruses in the patient’s sample. Hemagglutination is the agglutination (clumping) together of erythrocytes (red blood cells). Many viruses produce surface proteins or spikes called hemagglutinins that can bind to receptors on the membranes of erythrocytes and cause the cells to agglutinate. Hemagglutination is observable without using the microscope, but this method does not always differentiate between infectious and noninfectious viral particles, since both can agglutinate erythrocytes.

To identify a specific pathogenic virus using hemagglutination, we must use an indirect approach. Proteins called antibodies, generated by the patient’s immune system to fight a specific virus, can be used to bind to components such as hemagglutinins that are uniquely associated with specific types of viruses. The binding of the antibodies with the hemagglutinins found on the virus subsequently prevent erythrocytes from directly interacting with the virus. So when erythrocytes are added to the antibody-coated viruses, there is no appearance of agglutination; agglutination has been inhibited. We call these types of indirect assays for virus-specific antibodies hemagglutination inhibition (HAI) assays. HAI can be used to detect the presence of antibodies specific to many types of viruses that may be causing or have caused an infection in a patient even months or years after infection (see Figure 6). This assay is described in greater detail in Agglutination Assays.

Figure 6. This chart shows the possible outcomes of a hemagglutination test. Row A: Erythrocytes do not bind together and will sink to the bottom of the well plate; this becomes visible as a red dot in the center of the well. Row B: Many viruses have hemagglutinins that causes agglutination of erythrocytes; the resulting hemagglutination forms a lattice structure that results in red color throughout the well. Row C: Virus-specific antibody, the viruses, and the erythrocytes are added to the well plate. The virus-specific antibodies inhibit agglutination, as can be seen as a red dot in the bottom of the well. (credit: modification of work by Centers for Disease Control and Prevention)

Think about It

  • What is the outcome of a positive HIA test?

Nucleic Acid Amplification Test

Nucleic acid amplification tests (NAAT) are used in molecular biology to detect unique nucleic acid sequences of viruses in patient samples. Polymerase chain reaction (PCR) is an NAAT used to detect the presence of viral DNA in a patient’s tissue or body fluid sample. PCR is a technique that amplifies (i.e., synthesizes many copies) of a viral DNA segment of interest. Using PCR, short nucleotide sequences called primers bind to specific sequences of viral DNA, enabling identification of the virus.

Reverse transcriptase-PCR (RT-PCR) is an NAAT used to detect the presence of RNA viruses. RT-PCR differs from PCR in that the enzyme reverse transcriptase (RT) is used to make a cDNA from the small amount of viral RNA in the specimen. The cDNA can then be amplified by PCR. Both PCR and RT-PCR are used to detect and confirm the presence of the viral nucleic acid in patient specimens.

HPV Scare

Michelle, a 21-year-old nursing student, came to the university clinic worried that she might have been exposed to a sexually transmitted disease (STD). Her sexual partner had recently developed several bumps on the base of his penis. He had put off going to the doctor, but Michelle suspects they are genital warts caused by HPV. She is especially concerned because she knows that HPV not only causes warts but is a prominent cause of cervical cancer. She and her partner always use condoms for contraception, but she is not confident that this precaution will protect her from HPV.

Michelle’s physician finds no physical signs of genital warts or any other STDs, but recommends that Michelle get a Pap smear along with an HPV test. The Pap smear will screen for abnormal cervical cells and the CPEs associated with HPV; the HPV test will test for the presence of the virus. If both tests are negative, Michelle can be more assured that she most likely has not become infected with HPV. However, her doctor suggests it might be wise for Michelle to get vaccinated against HPV to protect herself from possible future exposure.

  • Why does Michelle’s physician order two different tests instead of relying on one or the other?

Enzyme Immunoassay

Enzyme immunoassays (EIAs) rely on the ability of antibodies to detect and attach to specific biomolecules called antigens. The detecting antibody attaches to the target antigen with a high degree of specificity in what might be a complex mixture of biomolecules. Also included in this type of assay is a colorless enzyme attached to the detecting antibody. The enzyme acts as a tag on the detecting antibody and can interact with a colorless substrate, leading to the production of a colored end product. EIAs often rely on layers of antibodies to capture and react with antigens, all of which are attached to a membrane filter (see Figure 7). EIAs for viral antigens are often used as preliminary screening tests. If the results are positive, further confirmation will require tests with even greater sensitivity, such as a western blot or an NAAT. EIAs are discussed in more detail in EIAs and ELISAs.

Figure 7. Click to view larger image. Similar to rapid, over-the-counter pregnancy tests, EIAs for viral antigens require a few drops of diluted patient serum or plasma applied to a membrane filter. The membrane filter has been previously modified and embedded with antibody to viral antigen and internal controls. Antibody conjugate is added to the filter, with the targeted antibody attached to the antigen (in the case of a positive test). Excess conjugate is washed off the filter. Substrate is added to activate the enzyme-mediated reaction to reveal the color change of a positive test. (credit: modification of work by “Cavitri”/Wikimedia Commons)

Think about It

  • What typically indicates a positive EIA test?

Clinical Focus: Joaquim, Part 3

This example continues Joaquim’s story that started earlier on Viruses.

Along with the RT/PCR analysis, Joaquim’s saliva was also collected for viral cultivation. In general, no single diagnostic test is sufficient for antemortem diagnosis, since the results will depend on the sensitivity of the assay, the quantity of virions present at the time of testing, and the timing of the assay, since release of virions in the saliva can vary. As it turns out, the result was negative for viral cultivation from the saliva. This is not surprising to Joaquim’s doctor, because one negative result is not an absolute indication of the absence of infection. It may be that the number of virions in the saliva is low at the time of sampling. It is not unusual to repeat the test at intervals to enhance the chance of detecting higher virus loads.

  • Should Joaquim’s doctor modify his course of treatment based on these test results?

We’ll return to Joaquim’s example in later pages.

Key Concepts and Summary

  • Viral cultivation requires the presence of some form of host cell (whole organism, embryo, or cell culture).
  • Viruses can be isolated from samples by filtration.
  • Viral filtrate is a rich source of released virions.
  • Bacteriophages are detected by presence of clear plaques on bacterial lawn.
  • Animal and plant viruses are detected by cytopathic effects, molecular techniques (PCR, RT-PCR), enzyme immunoassays, and serological assays (hemagglutination assay, hemagglutination inhibition assay).

Multiple Choice

Which of the followings cannot be used to culture viruses?

  1. tissue culture
  2. liquid medium only
  3. embryo
  4. animal host

Which of the following tests can be used to detect the presence of a specific virus?

Which of the following is NOT a cytopathic effect?

  1. transformation
  2. cell fusion
  3. mononucleated cell
  4. inclusion bodies

Fill in the Blank

Viruses can be diagnosed and observed using a(n) _____________ microscope.

Cell abnormalities resulting from a viral infection are called _____________.

Think about It

  1. Briefly explain the various methods of culturing viruses.
  2. What are some characteristics of the viruses that are similar to a computer virus?
  3. Label the components indicated by arrows.

(credit: modification of work by American Society for Microbiology)

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Ranatra rod-shaped: a description of the appearance and biological development of a water bug

Prokaryotes are identified as gram-positive if they have a multiple layer matrix of peptidoglycan forming the cell wall. Crystal violet, the primary stain of the Gram stain procedure, is readily retained and stabilized within this matrix, causing gram-positive prokaryotes to appear purple under a brightfield microscope after Gram staining. For many years, the retention of Gram stain was one of the main criteria used to classify prokaryotes, even though some prokaryotes did not readily stain with either the primary or secondary stains used in the Gram stain procedure.

Advances in nucleic acid biochemistry have revealed additional characteristics that can be used to classify gram-positive prokaryotes, namely the guanine to cytosine ratios (G+C) in DNA and the composition of 16S rRNA subunits. Microbiologists currently recognize two distinct groups of gram-positive, or weakly staining gram-positive, prokaryotes. The class Actinobacteria comprises the high G+C gram-positive bacteria, which have more than 50% guanine and cytosine nucleotides in their DNA. The class Bacilli comprises low G+C gram-positive bacteria, which have less than 50% of guanine and cytosine nucleotides in their DNA.

Actinobacteria: High G+C Gram-Positive Bacteria

The name Actinobacteria comes from the Greek words for rays and small rod, but Actinobacteria are very diverse. Their microscopic appearance can range from thin filamentous branching rods to coccobacilli. Some Actinobacteria are very large and complex, whereas others are among the smallest independently living organisms. Most Actinobacteria live in the soil, but some are aquatic. The vast majority are aerobic. One distinctive feature of this group is the presence of several different peptidoglycans in the cell wall.

The genus Actinomyces is a much studied representative of Actinobacteria. Actinomyces spp. play an important role in soil ecology, and some species are human pathogens. A number of Actinomyces spp. inhabit the human mouth and are opportunistic pathogens, causing infectious diseases like periodontitis (inflammation of the gums) and oral abscesses. The species A. israelii is an anaerobe notorious for causing endocarditis (inflammation of the inner lining of the heart) (Figure 1).

Figure 1. (a) Actinomyces israelii (false-color scanning electron micrograph [SEM]) has a branched structure. (b) Corynebacterium diphtheria causes the deadly disease diphtheria. Note the distinctive palisades. (c) The gram-variable bacterium Gardnerella vaginalis causes bacterial vaginosis in women. This micrograph shows a Pap smear from a woman with vaginosis. (credit a: modification of work by “GrahamColm”/Wikimedia Commons; credit b: modification of work by Centers for Disease Control and Prevention; credit c: modification of work by Mwakigonja AR, Torres LM, Mwakyoma HA, Kaaya EE)

The genus Mycobacterium is represented by bacilli covered with a mycolic acid coat. This waxy coat protects the bacteria from some antibiotics, prevents them from drying out, and blocks penetration by Gram stain reagents (see Staining Microscopic Specimens). Because of this, a special acid-fast staining procedure is used to visualize these bacteria. The genus Mycobacterium is an important cause of a diverse group of infectious diseases. M. tuberculosis is the causative agent of tuberculosis, a disease that primarily impacts the lungs but can infect other parts of the body as well. It has been estimated that one-third of the world’s population has been infected with M. tuberculosis and millions of new infections occur each year. Treatment of M. tuberculosis is challenging and requires patients to take a combination of drugs for an extended time. Complicating treatment even further is the development and spread of multidrug-resistant strains of this pathogen.

Another pathogenic species, M. leprae, is the cause of Hansen’s disease (leprosy), a chronic disease that impacts peripheral nerves and the integrity of the skin and mucosal surface of the respiratory tract. Loss of pain sensation and the presence of skin lesions increase susceptibility to secondary injuries and infections with other pathogens.

Bacteria in the genus Corynebacterium contain diaminopimelic acid in their cell walls, and microscopically often form palisades, or pairs of rod-shaped cells resembling the letter V. Cells may contain metachromatic granules, intracellular storage of inorganic phosphates that are useful for identification of Corynebacterium. The vast majority of Corynebacterium spp. are nonpathogenic; however, C. diphtheria is the causative agent of diphtheria, a disease that can be fatal, especially in children (Figure 1b). C. diphtheria produces a toxin that forms a pseudomembrane in the patient’s throat, causing swelling, difficulty breathing, and other symptoms that can become serious if untreated.

The genus Bifidobacterium consists of filamentous anaerobes, many of which are commonly found in the gastrointestinal tract, vagina, and mouth. In fact, Bifidobacterium spp. constitute a substantial part of the human gut microbiota and are frequently used as probiotics and in yogurt production.

The genus Gardnerella, contains only one species, G. vaginalis. This species is defined as “gram-variable” because its small coccobacilli do not show consistent results when Gram stained (Figure 1c). Based on its genome, it is placed into the high G+C gram-positive group. G. vaginalis can cause bacterial vaginosis in women; symptoms are typically mild or even undetectable, but can lead to complications during pregnancy.

Table 1 summarizes the characteristics of some important genera of Actinobacteria. Additional information on Actinobacteria appears in Taxonomy of Clinically Relevant Microorganisms.

Table 1. Actinobacteria: High G+C Gram-Positive
Example Genus Microscopic Morphology Unique Characteristics
Actinomyces Gram-positive bacillus; in colonies, shows fungus-like threads (hyphae) Facultative anaerobes; in soil, decompose organic matter; in the human mouth, may cause gum disease
Arthrobacter Gram-positive bacillus (at the exponential stage of growth) or coccus (in stationary phase) Obligate aerobes; divide by “snapping,” forming V-like pairs of daughter cells; degrade phenol, can be used in bioremediation
Bifidobacterium Gram-positive, filamentous actinobacterium Anaerobes commonly found in human gut microbiota
Corynebacterium Gram-positive bacillus Aerobes or facultative anaerobes; form palisades; grow slowly; require enriched media in culture; C. diphtheriae causes diphtheria
Frankia Gram-positive, fungus-like (filamentous) bacillus Nitrogen-fixing bacteria; live in symbiosis with legumes
Gardnerella Gram-variable coccobacillus Colonize the human vagina, may alter the microbial ecology, thus leading to vaginosis
Micrococcus Gram-positive coccus, form microscopic clusters Ubiquitous in the environment and on the human skin; oxidase-positive (as opposed to morphologically similar S. aureus); some are opportunistic pathogens
Mycobacterium Gram-positive, acid-fast bacillus Slow growing, aerobic, resistant to drying and phagocytosis; covered with a waxy coat made of mycolic acid; M. tuberculosis causes tuberculosis; M. leprae causes leprosy
Nocardia Weakly gram-positive bacillus; forms acid-fast branches May colonize the human gingiva; may cause severe pneumonia and inflammation of the skin
Propionibacterium Gram-positive bacillus Aerotolerant anaerobe; slow-growing; P. acnes reproduces in the human sebaceous glands and may cause or contribute to acne
Rhodococcus Gram-positive bacillus Strict aerobe; used in industry for biodegradation of pollutants; R. fascians is a plant pathogen, and R. equi causes pneumonia in foals
Streptomyces Gram-positive, fungus-like (filamentous) bacillus Very diverse genus (>500 species); aerobic, spore-forming bacteria; scavengers, decomposers found in soil (give the soil its “earthy” odor); used in pharmaceutical industry as antibiotic producers (more than two-thirds of clinically useful antibiotics)

Think about It

  • What is one distinctive feature of Actinobacteria?

Low G+C Gram-positive Bacteria

The low G+C gram-positive bacteria have less than 50% guanine and cytosine in their DNA, and this group of bacteria includes a number of genera of bacteria that are pathogenic.

Clinical Focus: Sharnita, Part 3

Based on her symptoms, Sharnita’s doctor suspected that she had a case of tuberculosis. Although less common in the United States, tuberculosis is still extremely common in many parts of the world, including Nigeria. Sharnita’s work there in a medical lab likely exposed her to Mycobacterium tuberculosis, the bacterium that causes tuberculosis.

Sharnita’s doctor ordered her to stay at home, wear a respiratory mask, and confine herself to one room as much as possible. He also said that Sharnita had to take one semester off school. He prescribed isoniazid and rifampin, antibiotics used in a drug cocktail to treat tuberculosis, which Marsha was to take three times a day for at least three months.

  • Why did the doctor order Sharnita to stay home for three months?

We’ll conclude Sharnita’s example later in this page.

Clostridia

One large and diverse class of low G+C gram-positive bacteria is Clostridia. The best studied genus of this class is Clostridium. These rod-shaped bacteria are generally obligate anaerobes that produce endospores and can be found in anaerobic habitats like soil and aquatic sediments rich in organic nutrients. The endospores may survive for many years.

Figure 2. Clostridium difficile, a gram-positive, rod-shaped bacterium, causes severe colitis and diarrhea, often after the normal gut microbiota is eradicated by antibiotics. (credit: modification of work by Centers for Disease Control and Prevention)

Clostridium spp. produce more kinds of protein toxins than any other bacterial genus, and several species are human pathogens. C. perfringens is the third most common cause of food poisoning in the United States and is the causative agent of an even more serious disease called gas gangrene. Gas gangrene occurs when C. perfringens endospores enter a wound and germinate, becoming viable bacterial cells and producing a toxin that can cause the necrosis (death) of tissue. C. tetani, which causes tetanus, produces a neurotoxin that is able to enter neurons, travel to regions of the central nervous system where it blocks the inhibition of nerve impulses involved in muscle contractions, and cause a life-threatening spastic paralysis. C. botulinum produces botulinum neurotoxin, the most lethal biological toxin known. Botulinum toxin is responsible for rare but frequently fatal cases of botulism. The toxin blocks the release of acetylcholine in neuromuscular junctions, causing flaccid paralysis. In very small concentrations, botulinum toxin has been used to treat muscle pathologies in humans and in a cosmetic procedure to eliminate wrinkles. C. difficile is a common source of hospital-acquired infections (Figure 2) that can result in serious and even fatal cases of colitis (inflammation of the large intestine). Infections often occur in patients who are immunosuppressed or undergoing antibiotic therapy that alters the normal microbiota of the gastrointestinal tract. Taxonomy of Clinically Relevant Microorganisms lists the genera, species, and related diseases for Clostridia.

Lactobacillales

The order Lactobacillales comprises low G+C gram-positive bacteria that include both bacilli and cocci in the genera Lactobacillus, Leuconostoc, Enterococcus, and Streptococcus. Bacteria of the latter three genera typically are spherical or ovoid and often form chains.

Streptococcus, the name of which comes from the Greek word for twisted chain, is responsible for many types of infectious diseases in humans. Species from this genus, often referred to as streptococci, are usually classified by serotypes called Lancefield groups, and by their ability to lyse red blood cells when grown on blood agar.

S. pyogenes belongs to the Lancefield group A, β-hemolytic Streptococcus. This species is considered a pyogenic pathogen because of the associated pus production observed with infections it causes (Figure 3). S. pyogenes is the most common cause of bacterial pharyngitis (strep throat); it is also an important cause of various skin infections that can be relatively mild (e.g., impetigo) or life threatening (e.g., necrotizing fasciitis, also known as flesh eating disease), life threatening.

Figure 3. (a) A gram-stained specimen of Streptococcus pyogenes shows the chains of cocci characteristic of this organism’s morphology. (b) S. pyogenes on blood agar shows characteristic lysis of red blood cells, indicated by the halo of clearing around colonies. (credit a, b: modification of work by American Society for Microbiology)

The nonpyogenic (i.e., not associated with pus production) streptococci are a group of streptococcal species that are not a taxon but are grouped together because they inhabit the human mouth. The nonpyogenic streptococci do not belong to any of the Lancefield groups. Most are commensals, but a few, such as S. mutans, are implicated in the development of dental caries.

S. pneumoniae (commonly referred to as pneumococcus), is a Streptococcus species that also does not belong to any Lancefield group. S. pneumoniae cells appear microscopically as diplococci, pairs of cells, rather than the long chains typical of most streptococci. Scientists have known since the 19th century that S. pneumoniae causes pneumonia and other respiratory infections. However, this bacterium can also cause a wide range of other diseases, including meningitis, septicemia, osteomyelitis, and endocarditis, especially in newborns, the elderly, and patients with immunodeficiency.

Bacilli

The name of the class Bacilli suggests that it is made up of bacteria that are bacillus in shape, but it is a morphologically diverse class that includes bacillus-shaped and cocccus-shaped genera. Among the many genera in this class are two that are very important clinically: Bacillus and Staphylococcus.

Bacteria in the genus Bacillus are bacillus in shape and can produce endospores. They include aerobes or facultative anaerobes. A number of Bacillus spp. are used in various industries, including the production of antibiotics (e.g., barnase), enzymes (e.g., alpha-amylase, BamH1 restriction endonuclease), and detergents (e.g., subtilisin).

Two notable pathogens belong to the genus Bacillus. B. anthracis is the pathogen that causes anthrax, a severe disease that affects wild and domesticated animals and can spread from infected animals to humans. Anthrax manifests in humans as charcoal-black ulcers on the skin, severe enterocolitis, pneumonia, and brain damage due to swelling. If untreated, anthrax is lethal. B. cereus, a closely related species, is a pathogen that may cause food poisoning. It is a rod-shaped species that forms chains. Colonies appear milky white with irregular shapes when cultured on blood agar (Figure 4). One other important species is B. thuringiensis. This bacterium produces a number of substances used as insecticides because they are toxic for insects.

Figure 4. (a) In this gram-stained specimen, the violet rod-shaped cells forming chains are the gram-positive bacteria Bacillus cereus. The small, pink cells are the gram-negative bacteria Escherichia coli. (b) In this culture, white colonies of B. cereus have been grown on sheep blood agar. (credit a: modification of work by “Bibliomaniac 15″/Wikimedia Commons; credit b: modification of work by Centers for Disease Control and Prevention)

Figure 5. This SEM of Staphylococcus aureus illustrates the typical “grape-like” clustering of cells. (credit: modification of work by Centers for Disease Control and Prevention)

The genus Staphylococcus also belongs to the class Bacilli, even though its shape is coccus rather than a bacillus. The name Staphylococcus comes from a Greek word for bunches of grapes, which describes their microscopic appearance in culture (Figure 5). Staphylococcus spp. are facultative anaerobic, halophilic, and nonmotile. The two best-studied species of this genus are S. epidermidis and S. aureus.

S. epidermidis, whose main habitat is the human skin, is thought to be nonpathogenic for humans with healthy immune systems, but in patients with immunodeficiency, it may cause infections in skin wounds and prostheses (e.g., artificial joints, heart valves). S. epidermidis is also an important cause of infections associated with intravenous catheters. This makes it a dangerous pathogen in hospital settings, where many patients may be immunocompromised.

Strains of S. aureus cause a wide variety of infections in humans, including skin infections that produce boils, carbuncles, cellulitis, or impetigo. Certain strains of S. aureus produce a substance called enterotoxin, which can cause severe enteritis, often called staph food poisoning. Some strains of S. aureus produce the toxin responsible for toxic shock syndrome, which can result in cardiovascular collapse and death.

Many strains of S. aureus have developed resistance to antibiotics. Some antibiotic-resistant strains are designated as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). These strains are some of the most difficult to treat because they exhibit resistance to nearly all available antibiotics, not just methicillin and vancomycin. Because they are difficult to treat with antibiotics, infections can be lethal. MRSA and VRSA are also contagious, posing a serious threat in hospitals, nursing homes, dialysis facilities, and other places where there are large populations of elderly, bedridden, and/or immunocompromised patients. Taxonomy of Clinically Relevant Microorganisms lists the genera, species, and related diseases for bacilli.

Mycoplasmas

Although Mycoplasma spp. do not possess a cell wall and, therefore, are not stained by Gram-stain reagents, this genus is still included with the low G+C gram-positive bacteria. The genus Mycoplasma includes more than 100 species, which share several unique characteristics. They are very small cells, some with a diameter of about 0.2 μm, which is smaller than some large viruses. They have no cell walls and, therefore, are pleomorphic, meaning that they may take on a variety of shapes and can even resemble very small animal cells. Because they lack a characteristic shape, they can be difficult to identify. One species, M. pneumoniae, causes the mild form of pneumonia known as “walking pneumonia” or “atypical pneumonia.” This form of pneumonia is typically less severe than forms caused by other bacteria or viruses.

Table 3 summarizes the characteristics of notable genera low G+C Gram-positive bacteria.

Table 3. Bacilli: Low G+C Gram-Positive Bacteria
Example Genus Microscopic Morphology Unique Characteristics
Bacillus Large, gram-positive bacillus Aerobes or facultative anaerobes; form endospores; B. anthracis causes anthrax in cattle and humans, B. cereus may cause food poisoning
Clostridium Gram-positive bacillus Strict anaerobes; form endospores; all known species are pathogenic, causing tetanus, gas gangrene, botulism, and colitis
Enterococcus Gram-positive coccus; forms microscopic pairs in culture (resembling Streptococcus pneumoniae) Anaerobic aerotolerant bacteria, abundant in the human gut, may cause urinary tract and other infections in the nosocomial environment
Lactobacillus Gram-positive bacillus Facultative anaerobes; ferment sugars into lactic acid; part of the vaginal microbiota; used as probiotics
Leuconostoc Gram-positive coccus; may form microscopic chains in culture Fermenter, used in food industry to produce sauerkraut and kefir
Mycoplasma The smallest bacteria; appear pleomorphic under electron microscope Have no cell wall; classified as low G+C Gram-positive bacteria because of their genome; M. pneumoniae causes “walking” pneumonia
Staphylococcus Gram-positive coccus; forms microscopic clusters in culture that resemble bunches of grapes Tolerate high salt concentration; facultative anaerobes; produce catalase; S. aureus can also produce coagulase and toxins responsible for local (skin) and generalized infections
Streptococcus Gram-positive coccus; forms chains or pairs in culture Diverse genus; classified into groups based on sharing certain antigens; some species cause hemolysis and may produce toxins responsible for human local (throat) and generalized disease
Ureaplasma Similar to Mycoplasma Part of the human vaginal and lower urinary tract microbiota; may cause inflammation, sometimes leading to internal scarring and infertility

Think about It

  • Name some ways in which streptococci are classified.
  • Name one pathogenic low G+C gram-positive bacterium and a disease it causes.

Clinical Focus: Sharnita, Resolution

This example concludes Sharnita’s story that started in Prokaryote Habitats, Relationships, and Microbiomes, Proteobacteria, and above

Marsha’s sputum sample was sent to the microbiology lab to confirm the identity of the microorganism causing her infection. The lab also performed antimicrobial susceptibility testing (AST) on the sample to confirm that the physician has prescribed the correct antimicrobial drugs.

Figure 6. M. tuberculosis grows on Löwenstein-Jensen (LJ) agar in distinct colonies. (credit: Centers for Disease Control and Prevention)

Direct microscopic examination of the sputum revealed acid-fast bacteria (AFB) present in Marsha’s sputum. When placed in culture, there were no signs of growth for the first 8 days, suggesting that microorganism was either dead or growing very slowly. Slow growth is a distinctive characteristic of M. tuberculosis.

After four weeks, the lab microbiologist observed distinctive colorless granulated colonies (Figure 6). The colonies contained AFB showing the same microscopic characteristics as those revealed during the direct microscopic examination of Marsha’s sputum. To confirm the identification of the AFB, samples of the colonies were analyzed using nucleic acid hybridization, or direct nucleic acid amplification (NAA) testing. When a bacterium is acid-fast, it is classified in the family Mycobacteriaceae. DNA sequencing of variable genomic regions of the DNA extracted from these bacteria revealed that it was high G+C. This fact served to finalize Marsha’s diagnosis as infection with M. tuberculosis. After nine months of treatment with the drugs prescribed by her doctor, Marsha made a full recovery.

Biopiracy and Bioprospecting

In 1969, an employee of a Swiss pharmaceutical company was vacationing in Norway and decided to collect some soil samples. He took them back to his lab, and the Swiss company subsequently used the fungus Tolypocladium inflatum in those samples to develop cyclosporine A, a drug widely used in patients who undergo tissue or organ transplantation. The Swiss company earns more than $1 billion a year for production of cyclosporine A, yet Norway receives nothing in return—no payment to the government or benefit for the Norwegian people. Despite the fact the cyclosporine A saves numerous lives, many consider the means by which the soil samples were obtained to be an act of “biopiracy,” essentially a form of theft. Do the ends justify the means in a case like this?

Nature is full of as-yet-undiscovered bacteria and other microorganisms that could one day be used to develop new life-saving drugs or treatments. [1] Pharmaceutical and biotechnology companies stand to reap huge profits from such discoveries, but ethical questions remain. To whom do biological resources belong? Should companies who invest (and risk) millions of dollars in research and development be required to share revenue or royalties for the right to access biological resources?

Compensation is not the only issue when it comes to bioprospecting. Some communities and cultures are philosophically opposed to bioprospecting, fearing unforeseen consequences of collecting genetic or biological material. Native Hawaiians, for example, are very protective of their unique biological resources.

For many years, it was unclear what rights government agencies, private corporations, and citizens had when it came to collecting samples of microorganisms from public land. Then, in 1993, the Convention on Biological Diversity granted each nation the rights to any genetic and biological material found on their own land. Scientists can no longer collect samples without a prior arrangement with the land owner for compensation. This convention now ensures that companies act ethically in obtaining the samples they use to create their products.

Key Concepts and Summary

  • Gram-positive bacteria are a very large and diverse group of microorganisms. Understanding their taxonomy and knowing their unique features is important for diagnostics and treatment of infectious diseases.
  • Gram-positive bacteria are classified into high G+C gram-positive and low G+C gram-positive bacteria, based on the prevalence of guanine and cytosine nucleotides in their genome
  • Actinobacteria is the taxonomic name of the class of high G+C gram-positive bacteria. This class includes the genera Actinomyces, Arthrobacter, Corynebacterium, Frankia, Gardnerella, Micrococcus, Mycobacterium, Nocardia, Propionibacterium, Rhodococcus, and Streptomyces. Some representatives of these genera are used in industry; others are human or animal pathogens.
  • Examples of high G+C gram-positive bacteria that are human pathogens include Mycobacteriumtuberculosis, which causes tuberculosis; M. leprae, which causes leprosy (Hansen’s disease); and Corynebacteriumdiphtheriae, which causes diphtheria.
  • Clostridia spp. are low G+C gram-positive bacteria that are generally obligate anaerobes and can form endospores. Pathogens in this genus include C.perfringens (gas gangrene), C. tetani (tetanus), and C. botulinum (botulism).
  • Lactobacillales include the genera Enterococcus, Lactobacillus, Leuconostoc, and Streptococcus. Streptococcus is responsible for many human diseases, including pharyngitis (strep throat), scarlet fever, rheumatic fever, glomerulonephritis, pneumonia, and other respiratory infections.
  • Bacilli is a taxonomic class of low G+C gram-positive bacteria that include rod-shaped and coccus-shaped species, including the genera Bacillus and Staphylococcus. B. anthracis causes anthrax, B. cereus may cause opportunistic infections of the gastrointestinal tract, and S.aureus strains can cause a wide range of infections and diseases, many of which are highly resistant to antibiotics.
  • Mycoplasma spp. are very small, pleomorphic low G+C gram-positive bacteria that lack cell walls. M. pneumoniae causes atypical pneumonia.

Multiple Choice

Which of the following bacterial species is classified as high G+C gram-positive?

  1. Corynebacterium diphtheriae
  2. Staphylococcus aureus
  3. Bacillus anthracis
  4. Streptococcus pneumonia

Fill in the Blank

Streptococcus is the ________ of bacteria that is responsible for many human diseases.

One species of Streptococcus, S. pyogenes, is a classified as a ________ pathogen due to the characteristic production of pus in infections it causes.

Propionibacterium belongs to ________ G+C gram-positive bacteria. One of its species is used in the food industry and another causes acne.

Think about It

  1. Name and describe two types of S. aureus that show multiple antibiotic resistance.
  2. The microscopic growth pattern shown is characteristic of which genus of bacteria?

(credit: modification of work by Janice Haney Carr/Centers for Disease Control and Prevention)

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