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Question about microaerophilic parasite tolerance for oxygen

Question about microaerophilic parasite tolerance for oxygen


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Does anyone know what is the maximal concentration of oxygen that can be tolerated by the parasite Giardia intestinalis?


Short answer: 50-60 µM O2 is ok; 80 µM is toxic.

Giardia lamblia (aka intestinalis) is a microaerophilic intestinal parasite.

According to:

Lloyd et al. (2000) The microaerophilic flagellate Giardia intestinalis: oxygen and its reaction products collapse membrane potential and cause cytotoxicity. Microbiology 146: 3109 - 3118

Giardia colonises the upper intestine, and the oxygen concentration there has been measured at 60 µM. At oxygen concentrations up to 50 µM the organism can scavenge oxygen; above 80 µM oxygen inhibits the oxygen-scavenging process. In this paper the authors used 10% air (equivalent to 25 µM oxygen) to characterise oxygen toxicity.

The source of oxygen sensitivity was investigated by:

Li and Wang (2006) A likely molecular basis of the susceptibility of Giardia lamblia towards oxygen Molecular Microbiology 59: 202 - 211

These authors propose that the enzyme DT-diaphorase, previously suggested as a component of the oxygen detoxification system promotes the formation of reactive oxygen species which lead to death. Thus they found that overproduction increased sensitivity to oxygen, whereas a knock-down decreased sensitivity. This work does not, however, use controlled microaerophilic conditions.


Question about microaerophilic parasite tolerance for oxygen - Biology

Recommended courses: Neurobiology (BIOL 386), Cell Biology (BIOL 280) or Immunology (BIOL 310)

Microglia, which surveil the central nervous system, have become much better understood over the last decade, as we have gradually documented the role of receptor and ion channel function in their operation. Review our current understanding of ion channel function in microglia, especially in the context of microglia’s neuro-protective functions.

Boucsein C. et al. Purinergic receptors on microglial cells: functional expression in acute brain slices and modulation of microglial activation in vitro. Eur. J. Neurosci. 2003 17: 2267-2276.

Madry C. et al. Microglial ramification, surveillance, and interleukin-1β release are regulated by the two-pore domain K + channel THIK-1. Neuron. 2018 97: 299-312.

Schilling T. Eder C. Microglial K + channel expression in young adult and aged mice. Glia. 2015 63: 664-672.

2. Role of Predation (David Hougen-Eitzman)

Predation (broadly defined to include parasitism and infection by pathogens) is a central organizing force in many biological systems. Predators can be powerful actors that affect species population size and distribution, trophic dynamics, and natural selection. In the system of your choice, review the ecological or evolutionary effects of predation.

Recommended courses: Population Ecology (Bio 352), Ecosystem Ecology (Bio 321), Agroecology (Bio 215), or Evolution (Bio 350)

Buck, J.C., Weinstein, S. B., and Young, H.S. 2018. Ecological and evolutionary consequences of parasite avoidance. Trends in Ecology and Evolution 33:619-632.

Gaynor, K.M., Brown, J.S., Middleton, A.D., Power, M.E., and Brashares, J.S. 2019. Landscapes of fear: spatial patterns of risk perception and response. Trends in Ecology and Evolution 34:355-368.

Tomasetto, F., Tylianakis, J.M., Reale, M., Wratten, S., and Goldson, S.L. 2017. Intensified agriculture favors evolved resistance to biological control. Proceedings of the National Academy of Sciences. 114:3885-3890

Zhang, J. Qian, H., Girardello, M., Pellissier, V., Nielsen, S.E., and Svenning, J.C. 2018. Trophic interactions among vertebrate guilds and plants shape global patterns in species diversity. Proceedings of the Royal Society B-Biological Sciences 285:20180949

3. Non-canonical roles of RNA (Rou-Jia Sung)

In the central dogma, RNA is typically relegated to the “intermediary” role between DNA and protein. However, RNA is an incredibly dynamic and extraordinary biological macromolecule, capable of adopting complex three-dimensional shapes that allow it to access a variety of biological functions far beyond its canonical role as temporary information storage. These functions range from catalysis (ribozymes) to regulation (riboswitches, noncoding RNAs, circular RNAs) and epitranscriptomics (chemical modifications to RNA)!

For this question, explore a non-canonical role of RNA and consider the molecular mechanism(s) and outcomes (at both the cellular and organismal level) that can result from this novel functionality.

Recommended courses: BIOL 380 or CHEM 320 or BIOL 280

Li, X, et al. 2018. The biogenesis, functions, and challenges of circular RNAs. Molecular Cell, 71: 428-442.

Roundtree, IA, et al. 2017. Dynamic RNA modifications in gene expression regulation. Cell, 169: 1187-1200.

Breaker, RR. 2018. Riboswitches and translation control. Cold Spring Harbor Perspectives in Biology, 1-14.

Topp, S and Gallivan, JP. 2010. Emerging applications of riboswitches in chemical biology. ACS Chemical Biology, 5 (1): 139-148.

4. Bacterial cells (Raka Mitra)

The field of cell biology has historically underestimated numerous aspects of bacterial cells. While eukaryotic cells were known to have membrane bound organelles, cytoskeletal proteins and innate immune pathways, these features were typically understudied in bacteria. Now, we know that bacteria make vesicles to transport materials between membranes. We know that bacteria employ proteins that are similar to eukaryotic actin and tubulin. We understand that bacteria have defense systems, such as CRISPR, in order to protect themselves against viruses.

Choose a system that has been understudied in bacterial cells explore the mechanisms that control this process on a cellular, molecular and biochemical level. Contrast this system to well-known eukaryotic systems in order to reveal surprises and new insights to be gained by studying the bacterial counterparts.

Recommended courses: Cell Biology (BIO280) and/or Microbiology (BIOL234)

Hille, F., Richter, H., Wong, S.P., Bratovic, M., Ressel, S., and Charpentier, E. (2018). The Biology of CRISPR-Cas: Backward and Forward. Cell 172, 1239-1259.

Toyofuku, M., Nomura, N., and Eberl, L. (2019). Types and origins of bacterial membrane vesicles. Nat Rev Microbiol 17, 13-24.

Wagstaff, J., and Lowe, J. (2018). Prokaryotic cytoskeletons: protein filaments organizing small cells. Nat Rev Microbiol 16, 187-201.

5. Life in air or water (Mike Nishizaki) [DHE will also advise this question]

For organisms living in any environment, life is spent immersed in a fluid, either air or water. Not surprisingly, many biological processes are intimately tied to the behavior of those fluids (i.e., patterns of flow). For instance, the fluid environment serves as an important medium that facilitates dispersal (e.g., pollen, larvae), transports nutrients and gases (e.g., photosynthesis, respiration, calcification), and imposes physical forces affecting the shape, size, and behavior of organisms. Examine how adaptations to flow are linked to the manner in which organisms respond to environmental change, including climate change.

Recommended courses: Physiological Ecology, Population Ecology, and/or Environmental Animal Physiology

Cummins, C., Seale, M., Macente, A., Certini, D., Mastropaolo, E., Viola, I. M., & Nakayama, N. (2018). A separated vortex ring underlies the flight of the dandelion. Nature, 562(7727), 414.

Gaylord, B., Hodin, J., & Ferner, M. C. (2013). Turbulent shear spurs settlement in larval sea urchins. Proceedings of the National Academy of Sciences, 110(17), 6901-6906.

Katija, K., & Dabiri, J. O. (2009). A viscosity-enhanced mechanism for biogenic ocean mixing. Nature, 460(7255), 624.

Mass, T., Genin, A., Shavit, U., Grinstein, M., & Tchernov, D. (2010). Flow enhances photosynthesis in marine benthic autotrophs by increasing the efflux of oxygen from the organism to the water. Proceedings of the National Academy of Sciences, 107(6), 2527-2531.

Morgan, S. G., Shanks, A. L., Fujimura, A. G., Reniers, A. J., MacMahan, J., Griesemer, C. D., Jarvis, M. & Brown, J. (2016). Surfzone hydrodynamics as a key determinant of spatial variation in rocky intertidal communities. Proceedings of the Royal Society B: Biological Sciences, 283(1840), 20161017.

Shishido, C. M., Woods, H. A., Lane, S. J., Toh, M. W. A., Tobalske, B. W., & Moran, A. L. (2019). Polar gigantism and the oxygen–temperature hypothesis: a test of upper thermal limits to body size in Antarctic pycnogonids. Proceedings of the Royal Society B, 286(1900), 20190124.

Stocking, J. B., Laforsch, C., Sigl, R., & Reidenbach, M. A. (2018). The role of turbulent hydrodynamics and surface morphology on heat and mass transfer in corals. Journal of the Royal Society Interface, 15(149), 20180448.

Timerman, D., & Barrett, S. C. (2018). Divergent selection on the biomechanical properties of stamens under wind and insect pollination. Proceedings of the Royal Society B, 285(1893), 20182251.

6. Immunotherapy and the mechanisms of CAR T cell therapy (Debby Walser-Kuntz)

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Explore an aspect of current CAR T cell therapy, focusing on the signaling pathways, regulation of key surface molecule expression, and the challenges of determining CAR T cell efficacy in vivo. Your synopsis should explain the underlying immune mechanisms involved in the design and function of CAR T cells and address the role and impact of the specific phenotype of CAR T cells, structural variations in CARs, and T cell exhaustion.

Recommended courses: Immunology (Bio 310) or Virology (Bio 370)

Kansal, R. et al., 2019. Sustained B cell depletion by CD19-targeted CAR T cells is a highly effective treatment for murine lupus. Science Translational Medicine 11.

Leick, M. & M. Maus. 2019. CAR T cells beyond CD19, UnCAR-Ted territory. American Journal of Hematology 94: S34-S41.

Wagner, T. 2018. Quarter century of anti-HIV CAR T cells. Current HIV/AIDS Reports 15:147–154.

Zhang, Q. et al. 2018. Chimeric Antigen Receptor (CAR) Treg: A promising approach to inducing immunological tolerance. Frontiers in Immunology 9: 1-8.

7. Breaking Symmetry (Jennifer Wolff)

The establishment of each of the primary embryonic axes (anterior-poster, dorsal-ventral, and left-right) is key to the structure and function of living organisms. Formation of the embryonic anterior-posterior and dorsal-ventral axes is critical to survival in early embryos, and has been well studied by developmental biologists for over a century. In contrast, asymmetries on the left-right axis, such as the looping of digestive system and heart and “handedness” of nervous system structures, are somewhat subtle, with their molecular and cellular origins, until recently, less well understood.

Identify a tissue or organ that exhibits asymmetry on the left-right axis, and discuss how this asymmetry contributes to its function. Describe the molecular and cellular processes by which this asymmetry develops, tracing a path from initial symmetry breaking through later events such as asymmetric morphogenesis, cell division, and gene expression.

Recommended courses: BIOL240, BIOL280, BIOL342, or BIOL368

Desgrange, A., Le Garrec, J.-F., and Meilhac, S.M. (2018). Left-right asymmetry in heart development and disease: forming the right loop. Development 145, dev162776–19.

Duboc, V., Dufourcq, P., Blader, P., and Roussigné, M. (2015). Asymmetry of the Brain: Development and Implications. Annu. Rev. Genet. 49, 647–672.

Grimes, D.T., and Burdine, R.D. (2017). Left–Right Patterning: Breaking Symmetry to Asymmetric Morphogenesis. Trends in Genetics 33, 616–628.

Levin, M., Klar, A.J.S., and Ramsdell, A.F. (2016). Introduction to provocative questions in left–right asymmetry. Phil. Trans. R. Soc. B 371, 20150399–7.

Zhang, H.T., and Hiiragi, T. (2018). Symmetry Breaking in the Mammalian Embryo. Annu Rev Cell Dev Biol 34, 405–426.

8. Reproductive Physiology (Matt Rand)

Physiology is an essential mediator of the reproductive phenotype. The regulatory mechanisms of reproduction through the hypothalamo-pituitary-gonad axis are fairly well understood for vertebrates, including humans. However, a comprehensive understanding of reproductive biology should include the mechanisms responsible for the onset and cessation of reproductive events, whether typical or anomalous. Information on how reproduction is initiated, ended, or interrupted by normal physiological means, environmental perturbations, or pathologies, is less well understood.

Choose a reproductive function or anomaly (e.g. sexual maturation, induced ovulation, early menarche, end-of-season gonadal quiescence, menopause, infertility, amenorrhea, reduced sperm count, etc.) and review the current literature in an attempt to understand the physiological mechanisms that mediate your chosen sample of the reproductive phenotype.

Recommended courses: Environmental Animal Physiology (252), Human Physiology (332), Human Reproduction and Sexuality (101), and/or Animal Development (342)

Angelopoulou, E., C. Quignon, L.J. Kriegsfeld, V. Simonneaux (2019) Functional Implications of RFRP-3 in the Central Control of Daily and Seasonal Rhythms in Reproduction. Front Endocrinol (Lausanne) 10183: 1-14.

Hart, R.J. (2016) Physiological aspects of female fertility: Role of the environment, modern lifestyle, and genetics. Physiol Rev 96: 873–909.

Leonardi, A., M. Cofini, D. Rigante, L. Lucchetti, C. Cipolla, L. Penta, and S. Esposito (2017) The Effect of Bisphenol A on Puberty: A Critical Review of the Medical Literature. Int J Environ Res Public Health 141044:1-20.


Background

The diplomonads are a diverse assemblage of parasitic and free-living eukaryotes that occupy low-oxygen environments. The best-studied diplomonad is the human parasite Giardia instestinalis which annually infects 280 million people worldwide [1]. This non-invasive, microaerophilic parasite colonizes the upper small intestine and causes the disease, giardiasis, which manifests in humans as an acute diarrhea that can develop to a chronic stage [2]. The life cycle comprises two main stages: the trophozoite, the actively replicating symptomatic stage, and the cyst, the environmentally resistant infective stage shed in host stools [2, 3]. Another lineage of parasitic diplomonads includes Spironucleus species known to infect a wide array of animals including birds, primates, and mice [4,5,6]. Some Spironucleus species infect piscine species where they cause numerous pathologies (e.g., skin lesions, anorexia, lethargy, abnormal swimming behaviors, and reclusiveness) detrimental to healthy fish rearing and are thus a growing concern for global aquaculture and ornamental fish industries [7,8,9,10]. The “salmon killer” Spironucleus salmonicida is known to infect the digestive tract of farmed Atlantic salmon, Arctic charr, and Chinook salmon (reviewed in [10]). Spironucleus outbreaks in farmed Atlantic salmon populations cause significant damage to the aquaculture economy, and the only treatment for spironucleosis, metronidazole, was banned in Europe in the late 1990s [11] due to its carcinogenic activity [12]. Therefore, advancing our current understanding of this parasite’s biology is essential for developing alternative treatment strategies and to block transmission cycles.

One feature that sets Spironucleus species apart from G. intestinalis, and most other intestinal protozoan parasites, is its ability to cause systemic infections. While the particular lifecycle is unknown for S. salmonicida, some details can be predicted from observations of close relatives. Cysts have been described in some Spironucleus species [5, 13] however, no formal molecular characterizations have been reported. Therefore, the trigger and site of encystation is unknown in piscine Spironucleus species. Encystation might occur in the gut and exit the host via the feces favoring a fecal-oral transmission model. Alternatively, encystation could occur on external lesions and is thus transferred between fish in close contact, favoring a skin-oral or skin-gill transmission model. Trophozoites have been observed in the gut, feces, skin, organs, and gills of fish infected with Spironucleus species and are the primary life stage observed under laboratory conditions [10, 14]. Once inside the host, the trophozoites are predicted to asexually reproduce in the gut and, in some cases, invade the host’s mucosa to enter the blood stream leading to systemic infections.

The ability of S. salmonicida to invade and persist in oxygenated tissues following gut colonization is particularly interesting given that it is classically viewed as an anaerobe. Indeed, molecular studies of S. salmonicida revealed a suite of oxygen-sensitive enzymes typically associated with anaerobiosis, some of which function in the mitochondrion-related organelles of S. salmonicida [15]. These “hydrogenosomes” have completely lost oxidative phosphorylation and instead rely exclusively on substrate-level phosphorylation to generate ATP [15]. Glucose- or amino acid-derived pyruvate is oxidized to acetyl-CoA via Pyruvate: ferredoxin oxidoreductase (PFO), and electrons are transferred via a Ferredoxin (FER) and [FeFe]-hydrogenase (HYD) ultimately reducing protons to hydrogen. The acetyl-CoA generated in this reaction can be further processed to acetate yielding one molecule of ATP via acetyl-CoA synthetase (ACS). Three accessory maturase proteins (HYD E,F,G) also function in the MRO to promote proper assembly of the Fe-S cluster of HYD. A similar pathway functions in the cytoplasm of S. salmonicida however, the electron carrier between PFO and HYD and also the proteins necessary in HYD maturation are currently unknown. In G. intestinalis, these pathways are exclusively localized to the cytoplasm.

While in the blood and tissues of its host, S. salmonicida is exposed to higher oxygen levels than those in the gut. Yet, the parasite does not encode proteins related to traditional oxygen defense pathways (e.g., superoxide dismutase, catalase, glutathione metabolism) [16, 17]. It is currently unclear how the organism adapts and thrives in oxygenated tissues throughout its life cycle. To investigate how this parasite tolerates oxidative stress, we performed transcriptional profiling of cultured S. salmonicida exposed to oxygen or anti-oxidant depletion. We observed that a large portion of the transcriptome is in fact upregulated in response to oxygen and anti-oxidant deprivation suggesting that this parasite employs a variety of strategies (e.g., oxygen clearance, iron-sequestration, and cysteine metabolism) to thrive in different oxygen tensions. Importantly, we show that some of the genes responsive to oxidative stress were transferred to S. salmonicida from bacterial donors via lateral gene transfer (LGT), thereby showcasing the role of LGT on the evolution of a eukaryote.


Use the following information to answer questions 20 and 21:

a team of ecologists measured the salinity of the water in an estuary at various distances from the river mouth. they also sampled populations of two species of clam worm, nereis occidentalis and neanthes succinea, at each point. the results are tabulated in the following table.

nereis occidentalis
(no. per 100 cm2)

neanthes succinea
(no. per 100 cm2)

distance from the river
mouth (meters)

20. which of the following hypotheses is most valuable in explaining the trends seen in the data?

a. nereis occidentalis outcompetes neanthes succinea at salinities under 14%.
b. neanthes succinea reproduces most rapidly in areas at least 20 meters from the river mouth.
c. both neanthes succinea and nereis occidentalis can survive at any salinity level.
d. nereis occidentalis is more resistant to salinity than neanthes succinea.

21. if the ecologists were to take a population sample of neanthes succinea in an area 65 meters from the river mouth with 30% salinity, what do you infer that they would find?

a. 40-50 individuals
b. 10-20 individuals
c. fewer than 5 individuals
d. more than 50 individuals

22. some coral reefs off the east coast of south america are starting to die off. satellite images reveal great plumes of sediment washing out of the mouths of rivers. what can you conclude, if anything, about the relationship between these two factors?

a. although the two factors coincide, they are not likely to be related.
b. deforestation on land results in lower oxygen levels in the atmosphere, which stresses the reefs.
c. deforestation on land allows erosion to wash away topsoil, creating sediment which smothers the reefs.
d. burning of the rain forest increases atmospheric carbon dioxide to levels toxic to reefs.

23. what is the distinction between a range of tolerance and limiting factors?

a. limiting factors are biotic or abiotic factors that limit the growth of a species, while the range of tolerance defines the set of conditions in which an organism can survive.
b. the range of tolerance defines biotic or abiotic factors that limit the growth of a species, while limiting factors define the set of conditions in which an organism can survive.
c. limiting factors and zones of tolerance are two terms for the same concepts about species survival under various environmental conditions.
d. limiting factors are biotic features only, such as interactions with other life forms, that limit a species, while range of tolerance is based only on abiotic conditions.

24. which of the following does not affect the spatial distribution of a population?

a. the carrying capacity of a population
b. the distribution of food and other resources
c. abiotic conditions such as rainfall and sunlight
d. the existence of predators or parasites

25. which of the following statements is correct?

a. population size of predators increases when their prey is scarce.
b. competition for resources is density-independent when food is plentiful.
c. disease is density-dependent because transmission occurs more easily when a population is large.
d. a change in average temperature is a density-dependent factor because fewer organisms can acclimate to variations in temperature.

26. how does the logistic model of population growth differ from the exponential model?

a. the exponential model shows a restricted growth rate.
b. the logistic model considers the environment’s carrying capacity.
c. the graph of the exponential model is s shaped.
d. the graph of the logistic model has a longer lag phase.

27. a fruit fly that has a short life span and produces many offspring can be classified into which reproductive strategy?

a. r-strategist
b. k-strategist
c. a carrying-capacity strategist
d. a logistic strategist

28. which characteristic is typical of a k-strategist?

a. short life span
b. generally a small organism
c. produces many offspring
d. lengthy parental care

29. which of the following methods might be used to decrease the rate of approach to carrying capacity by the developed world?

a. increase birthrate
b. decrease death rate
c. decrease resource use
d. decrease emigration


Question about microaerophilic parasite tolerance for oxygen - Biology

Article Summary:

Oxygen requirements of different bacteria

Almost all plants and animals are dependent upon supply of atmospheric oxygen which is unlikely in bacteria. Depending upon the oxygen need in bacteria, they are classified into 4 groups: strict or obligate aerobic that grow only in the presence of oxygen. Facultative anaerobic bacteria grow both in presence and absence of free oxygen. Some bacteria are microaerophilic which grow best in the presence of low concentration of molecular oxygen. However, not all bacteria require oxygen for growth. Strict or obligate anaerobic bacteria can grow only in the absence of oxygen.

Amount of oxygen required is different for growth and for other metabolic activities. Aerobic bacteria requires large surface growth area and exposure to contact available atmospheric oxygen, therefore under in vitro conditions, they are grown on shallow agar plates. Broth culturing of such bacteria is always accompanied by shaking and bubbling by spargers or baffles. Both of these mechanical actions are important during fermentation reactions to increase the availability and consumption of oxygen to growing aerobes. For the cultivation of anaerobic bacteria special techniques are employed which are meant to exclude atmospheric oxygen from growth medium. For this purpose, prereduced growth media contained with reducing agents like thioglycollate, formaldehyde, and sulfoxalate or cysteine hydrochloride which effectively absorb molecular oxygen are employed. Mechanical removal of oxygen by various means from an enclosed vessel containing tubes or plates of inoculated medium is another option. In one method, air is pumped out of vessel and replaced by gases like N2, helium or mixture of CO2 and N2. Burning of a candle to utilize oxygen present in the growth vessel is also one of the simplest ways to create oxygen free atmosphere. Addition of pyrogallol over the plug followed by rubber corking of test tube is used for slant cultivation of anaerobic bacteria. Anaerobic gaspak jars are routinely employed in various laboratories to cultivate anaerobes like Clostridium, Bacteroids and microaerophilic bacteria like Borrelia, Streptococci or Campylobacter.

Role of oxygen: Oxygen is elemental constituent of water and organic compounds. Obligatory aerobic bacteria are dependent on aerobic respiration for fulfillment of their energetic needs wherein molecular oxygen functions as terminal electron acceptor or oxidising agent. Anaerobic bacteria do not obtain energy by using molecular oxygen. In metabolic terms, facultative anaerobic bacteria can use oxygen as terminal oxidising agent only when available but can also obtain energy in its absence by fermentative reactions such as that in all enterobacteria. Some of them are not sensitive to presence of oxygen and hence have exclusively fermentative energy yielding metabolism lactic acid bacteria are representative of such fermentative metabolism. Microaerophilic bacteria grow best at oxygen concentration of 0.2atm pressure and possess enzymes that are inactivated during strong oxidising conditions, hence functional only at low oxygen pressure. Oxygen is also co-substrate for oxygenase enzymes that catalyze degradation of aromatic and alkane compounds. Oxygenative cleavage of aromatics is very useful for their dissimilation. Oxidative dissimilation of recalcitrant pollutant aromatics is hence one of the potential requirements for their efficient biodegradation. Oxygenases also mediate sterol and unsaturated fatty acid synthesis. They catalyze direct addition of one or two oxygen atoms to organic substrate compounds.

Oxygen toxicity: The role of nodulation in nitrogen fixing species of Rhizobium strains was understood only when researchers came to know about oxygen toxicity. Biological nitrogen fixation is catalyzed by nitrogenase enzyme system which is very sensitive to the presence of oxygen. It is readily inactivated by presence of molecular oxygen halting the vital process of fixation of atmospheric nitrogen. Similarly, hydrogen utilizing reactions catalyzed by enzyme hydrogenase accompanying the nitrogen fixation are also inhibited by oxygen. If oxygen is found at high concentration greater than atmosphere it can be toxic to aerobic bacteria oxygenases of aerobes are irreversibly denatured by exposure to oxygen. Therefore, bacteria especially aerobic, possessing oxygen sensitive enzymes have developed special mechanisms to protect functional enzymes from oxygen inactivation. Some mechanisms include high respiration rate, heterocyst formation, and synthesis of exopolysaccharides or nodulation. Enzymes like superoxide dismutase (SOD), peroxidase, and catalase are also used by these bacteria as shield against toxic forms of oxygen like superoxide radical (O2.-), hydrogen peroxide (H2O2) and hydroxyl radical (OH.). Lactic acid bacteria that don't possess catalase degrade H2O2 by peroxidase to H2O. Lethal accumulation of superoxide is prevented by SOD which catalyses its conversion to O2 and H2O2. Oxidations of flavoproteins by O2 results in the formation of toxic compounds like superoxide radicals. Oxygen is also toxic to anaerobic bacteria as they do not contain SOD (very little if present) or catalase therefore they have adapted to fermentative metabolism whereby they avoid presence of oxygen. Oxygen in its nascent or singlet state (1O2) is very toxic and powerful oxidant. Toxic singlet state is generated when triplet state of photosensitizer reacts with oxygen during photo-oxidation, a process similar to the production of superoxide radical. Its toxicity is enhanced in presence of light and photosensitive pigments or photosensitizers. Carotenoid pigments quench this form of oxygen and protect the cell from photo oxidative death. The chlorophylls are powerful photosensitizers and hence carotenoids are always present alongwith chlorophylls. Nonphotosynthetic aerobic bacteria like Micrococcus and Serratia also produce carotenoids in cell membrane to nullify the effect of photosensitizer cytochromes during their growth in high light intensified environment. Knowledge of oxygen requirements of bacteria is critical factor not only for their cultivation and classification but it also represents ecological significance.

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Science Practice Challenge Questions

Combustion of carbohydrates, like in a fireplace, is a reduction-oxidation reaction in which the carbon atom is oxidized and the oxygen atom is reduced, producing water and carbon dioxide. Oxidative phosphorylation and glycolysis are also reduction-oxidation reactions that produce the same products. Explain the differences and similarities among these abiotic and biotic processes in terms of the changes in entropy and heat that contribute to the free energy extracted from chemical bonds, the spontaneity of each, and the role of catalysis.

A. [Extension] Living systems require free energy to carry out cellular functions, and employ various strategies to capture, use, and store free energy. Explain the advantage that the higher energy efficiency per kg of the Krebs cycle provides to you compared to a metabolism based on glycolysis alone. Your explanation should make use of all the following facts:

  • ΔG for glycolysis is -135kJ per mole of glucose
  • ΔG for aerobic respiration is -2880kJ per mole glucose
  • the basal metabolic rate of mammals is often represented as -300kJ/day • m 0.75
  • the molar mass of glucose is 180 g/mole

B. Explain the bioenergetic difference between aerobic and anaerobic respiration in terms of the difference between free-energy production and power. Your explanation should make use of all the following facts:

  • power is the rate of free-energy production
  • cancer cells derive most of their free energy from glycolysis
  • enzymes of the citric acid (Kreb’s) cycle form coordinate complexes on the cytoskeleton within the mitochondria

C. The life cycle of the human parasite Trypanosoma brucei is divided between the body of the tsetse fly and the human blood stream. The parasite causes “sleeping sickness” in Sub-Saharan Africa. Within the human bloodstream, the parasite depends on glycolysis, with enzymes compartmentalized in a membrane-bound organelle called the glycosome. In the insect host, the parasite utilizes glycolysis as well as substrate-level and oxidative phosphorylation. Explain the advantage of a life cycle in the human host that employs anaerobic respiration with a rate of free-energy production that is enhanced by compartmentalization in the glycosome and a life cycle in the insect host that is aerobic.

D. Predict the advantages of a biological system that uses both glycolysis and oxidative phosphorylation. Your prediction should make use of all the following facts:

  • signaling can be used to detect low-oxygen environments and to regulate response
  • some cells, such as muscle and blood cells, must function in both low- and high-oxygen environments
  • glycolysis is reversible
  • the citric acid cycle is not reversible
  • thermoregulation is needed for homeostasis

Dinitrophenol (DNP) was used in the manufacture of munitions in World War I. In the 1930s, it was used as a weight loss drug. Use in the U.S. cannot be regulated by the FDA because DNP is considered a dietary supplement. Attempts to ban the drug in the U.K. following the death of four users in 2015 failed in Parliament. DNP is a small molecule that is soluble in the mitochondrial inner membrane. The hydroxyl group reversibly dissociates a proton.

A. Predict the effect of DNP on the electrochemical gradient across the inner mitochondrial membrane.

B. Explain how DNP can be used to reduce weight.

C. The effects of DNP can be reversed by administering glucose. However, treatment with a combination of glucose and 2-deoxyglucose, which is an inhibitor of glycolysis, does not reverse the effects of DNP. Explain, in terms of the products of glycolysis, why this reversal of the effects of DNP was unexpected. (Hint: It might be useful to review the reactants and products of glycolysis.)

D. Obesity correlates with an epidemic of other health issues, such as elevated blood pressure, heart disease, and diabetes II. A slow-release form of DNP (CRMP) is patented. With slow-release technology, a drug can be delivered in small doses over time from a pill whose matrix limits solubility. A simple but nonscientific question that can be raised is: Will a slow-release drug retard progress toward behavioral changes that can reduce the magnitude of this epidemic? Scientific questions can be pursued by testing the outcomes predicted by possible answers. Refine this question for discussion in small groups. Be prepared to justify the merits of your question.

As shown in Figure 7.11, cyanide inhibits the electron transport chain by competing with O2 molecules for the cytochrome c oxidase heme group. Carbon monoxide (CO) has a similar effect. Both cyanide and carbon monoxide cause poisoning in victims of smoke inhalation.

A. Predict the effects of these poisons on the following properties of mitochondria just after exposure: the pH of the intermembrane space, the concentration of NADH, and the rate of production of ATP in the matrix. Justify your predictions.

B. Rotenone is a poison that blocks the transfer of electrons from Complex I of the electron transport chain to ubiquinone. Methylene blue is a molecule with many uses involving its reduction-oxidation properties. Recent studies show the effectiveness of methylene blue in increasing the body’s metabolic rate and as a treatment for Alzheimer’s patients. The oxidized form of methylene blue is reduced by NADH, and its reduced form is oxidized by O2. Explain the use of methylene blue as an antidote for rotenone poisoning.

E. coli are enteric (gut-dwelling) facultative anaerobic bacteria. (Facultative anaerobes can grow either with or without free oxygen. Obligatory anaerobes grow only in the absence of free oxygen.) Researchers planned to grow cultures of E. coli under a range of conditions to model the transition from strictly anaerobic to aerobic respiration.

The oxygen content of atmospheres at constant total pressure will be controlled by volumes of nitrogen and oxygen gases. Ratios of volume, r = VO2/VN2 between 0 and 0.25 of shaken growth flasks can be measured in terms of optical density, which is the percent of transmission of light through a sample of the growing E. coli culture. A rule of thumb is that the range of strict anaerobes is when r < 0.01, and the boundary for aerobic respiration is when r = 0.05. A large number of flasks that can be constantly shaken at fixed temperature, and from which samples can be taken without atmospheric contamination, are available for this study.

These results of the experiment will be used to infer growth rates of E. coli along the entire 7.5 m length of the average human intestine (small intestine and large intestine), where the oxygen content varies from atmospheric to anaerobic conditions. The retention time of food in the small intestine, whose average length is 2.5 m, is approximately four hours. The retention time of food over the entire length of the intestine is between 24 and 72 hours.

A. Describe and apply a mathematical model that can be used to represent the variation of oxygen environments of a bacterium that is being transported with the food along the length of the intestine.

B. Design the experimental sampling times in terms of growth intervals of interest in this study: i) the time when the bacteria is passing the small-large intestine boundary ii) the time when the bacteria reaches the end of the large intestine and iii) the time when the bacterium reaches facultative anaerobic conditions, r < 0.05.

C. Sketch a graph that predicts the distribution of aerobic, facultative anaerobic and obligatory anaerobic bacteria along the length of the entire intestine based on these parameters. Keep in mind that anaerobes have a lower respiration rate.

White snakeroot is a plant that contains chemicals that deactivate the enzyme lactate dehydrogenase. Humans who consume milk from cows or goats that eat white snakeroot can become ill. Symptoms of milk poisoning include vomiting, abdominal pain, and tremors, which become worse after exercise. Beyond childhood, most people do not express the enzyme lactase that catalyzes the breakdown of lactose into glucose and galactose. Consumption of milk can produce symptoms similar to those of milk poisoning. After a period of consumption of dairy foods, though, prebiotic adaptation (changes in the microbes in the intestine) imparts lactose tolerance. Since dairy foods are a valuable source of calcium, proteins, and vitamin D, considerable research has been conducted to characterize adaptation.

Explain the similarities and differences between the effect of milk poisoning by white snakeroot and lactose intolerance, and the possibility of prebiotic adaptation for each.


THE PROCEDURES

Thioglycollate broth

  1. The thioglycollate broth should be either boiled first before inoculation OR recently made so that the oxygen content is very low. (Your instructor will tell you if it needs to be boiled).
  2. Inoculate a tube of thioglycollate broth with your unknown bacterium: make sure that the loop or needle goes down to the BOTTOM of the broth (do not get metal holder in the sterile broth).
  3. Incubate at 25 or 37 degrees C as directed.

TSA plates in 3 different oxygen environments

  1. Label 3 plates for the table---candle jar, ambient air, and GasPak anaerobic jar.
  2. Divide the 3 plates into sections, one for each organism&mdashyour unknown, the strict aerobe, the strict anaerobe.
  3. Inoculate the section by streaking a straight line or a zigzag (as seen below). HOWEVER, be sure that you inoculate all 3 plates using the same technique.
  4. Be sure that the jar has a methylene blue indicator strip (seen above) inside. The methylene blue is blue when oxidized, but colorless when reduced. Before the jar is opened, the strip should be checked to make sure that it is COLORLESS.
  5. Incubate at 30 or 37 degrees C

Gammaproteobacteria

The most diverse class of gram-negative bacteria is Gammaproteobacteria, and it includes a number of human pathogens. For example, a large and diverse family, Pseudomonaceae, includes the genus Pseudomonas. Within this genus is the species P. aeruginosa, a pathogen responsible for diverse infections in various regions of the body. P. aeruginosa is a strictly aerobic, nonfermenting, highly motile bacterium. It often infects wounds and burns, can be the cause of chronic urinary tract infections, and can be an important cause of respiratory infections in patients with cystic fibrosis or patients on mechanical ventilators. Infections by P. aeruginosa are often difficult to treat because the bacterium is resistant to many antibiotics and has a remarkable ability to form biofilms. Other representatives of Pseudomonas include the fluorescent (glowing) bacterium P. fluorescens and the soil bacteria P. putida, which is known for its ability to degrade xenobiotics (substances not naturally produced or found in living organisms).

The Pasteurellaceae also includes several clinically relevant genera and species. This family includes several bacteria that are human and/or animal pathogens. For example, Pasteurella haemolytica causes severe pneumonia in sheep and goats. P. multocida is a species that can be transmitted from animals to humans through bites, causing infections of the skin and deeper tissues. The genus Haemophilus contains two human pathogens, H. influenzae and H. ducreyi. Despite its name, H. influenzae does not cause influenza (which is a viral disease). H. influenzae can cause both upper and lower respiratory tract infections, including sinusitis, bronchitis, ear infections, and pneumonia. Before the development of effective vaccination, strains of H. influenzae were a leading cause of more invasive diseases, like meningitis in children. H. ducreyi causes the STI known as chancroid.

The order Vibrionales includes the human pathogen Vibrio cholerae. This comma-shaped aquatic bacterium thrives in highly alkaline environments like shallow lagoons and sea ports. A toxin produced by V. cholerae causes hypersecretion of electrolytes and water in the large intestine, leading to profuse watery diarrhea and dehydration. V. parahaemolyticus is also a cause of gastrointestinal disease in humans, whereas V. vulnificus causes serious and potentially life-threatening cellulitis (infection of the skin and deeper tissues) and blood-borne infections. Another representative of Vibrionales, Aliivibrio fischeri, engages in a symbiotic relationship with squid. The squid provides nutrients for the bacteria to grow and the bacteria produce bioluminescence that protects the squid from predators (Figure (PageIndex<5>)).

Figure (PageIndex<5>): (a) Aliivibrio fischeri is a bioluminescent bacterium. (b) A. fischeri colonizes and lives in a mutualistic relationship with the Hawaiian bobtail squid (Euprymna scolopes). (credit a: modification of work by American Society for Microbiology credit b: modification of work by Margaret McFall-Ngai)

The genus Legionella also belongs to the Gammaproteobacteria. L. pneumophila, the pathogen responsible for Legionnaires disease, is an aquatic bacterium that tends to inhabit pools of warm water, such as those found in the tanks of air conditioning units in large buildings (Figure (PageIndex<6>)). Because the bacteria can spread in aerosols, outbreaks of Legionnaires disease often affect residents of a building in which the water has become contaminated with Legionella. In fact, these bacteria derive their name from the first known outbreak of Legionnaires disease, which occurred in a hotel hosting an American Legion veterans&rsquo association convention in Philadelphia in 1976.

Figure (PageIndex<6>): (a) Legionella pneumophila, the causative agent of Legionnaires disease, thrives in warm water. (b) Outbreaks of Legionnaires disease often originate in the air conditioning units of large buildings when water in or near the system becomes contaminated with L. pneumophila. (credit a: modification of work by Centers for Disease Control and Prevention)

Enterobacteriaceae is a large family of enteric (intestinal) bacteria belonging to the Gammaproteobacteria. They are facultative anaerobes and are able to ferment carbohydrates. Within this family, microbiologists recognize two distinct categories. The first category is called the coliforms, after its prototypical bacterium species, Escherichia coli. Coliforms are able to ferment lactose completely (i.e., with the production of acid and gas). The second category, noncoliforms, either cannot ferment lactose or can only ferment it incompletely (producing either acid or gas, but not both). The noncoliforms include some notable human pathogens, such as Salmonella spp., Shigella spp., and Yersinia pestis.

E. coli has been perhaps the most studied bacterium since it was first described in 1886 by Theodor Escherich (1857&ndash1911). Many strains of E. coli are in mutualistic relationships with humans. However, some strains produce a potentially deadly toxin called Shiga toxin, which perforates cellular membranes in the large intestine, causing bloody diarrhea and peritonitis (inflammation of the inner linings of the abdominal cavity). Other E. coli strains may cause traveler&rsquos diarrhea, a less severe but very widespread disease.

The genus Salmonella, which belongs to the noncoliform group of Enterobacteriaceae, is interesting in that there is still no consensus about how many species it includes. Scientists have reclassified many of the groups they once thought to be species as serotypes (also called serovars), which are strains or variations of the same species of bacteria. Their classification is based on patterns of reactivity by animal antisera against molecules on the surface of the bacterial cells. A number of serotypes of Salmonella can cause salmonellosis, characterized by inflammation of the small and the large intestine, accompanied by fever, vomiting, and diarrhea. The species S. enterobacterica (serovar typhi) causes typhoid fever, with symptoms including fever, abdominal pain, and skin rashes (Figure (PageIndex<7>)).

Figure (PageIndex<7>): Salmonella typhi is the causative agent of typhoid fever. (credit: Centers for Disease Control and Prevention)

Table (PageIndex<3>) summarizes the characteristics of important genera of Gammaproteobacteria. Table (PageIndex<3>): Class Gammaproteobacteria
Example Genus Microscopic Morphology Unique Characteristics
Beggiatoa Gram-negative bacteria disc-shaped or cylindrical Aquatic, live in water with high content of hydrogen disulfide can cause problems for sewage treatment
Enterobacter Gram-negative bacillus Facultative anaerobe cause urinary and respiratory tract infections in hospitalized patients implicated in the pathogenesis of obesity
Erwinia Gram-negative bacillus Plant pathogen causing leaf spots and discoloration may digest cellulose prefer relatively low temperatures (25&ndash30 °C)
Escherichia Gram-negative bacillus Facultative anaerobe inhabit the gastrointestinal tract of warm-blooded animals some strains are mutualists, producing vitamin K others, like serotype E. coli O157:H7, are pathogens E. coli has been a model organism for many studies in genetics and molecular biology
Hemophilus Gram-negative bacillus Pleomorphic, may appear as coccobacillus, aerobe, or facultative anaerobe grow on blood agar pathogenic species can cause respiratory infections, chancroid, and other diseases
Klebsiella Gram-negative bacillus appears rounder and thicker than other members of Enterobacteriaceae Facultative anaerobe, encapsulated, nonmotile pathogenic species may cause pneumonia, especially in people with alcoholism
Legionella Gram-negative bacillus Fastidious, grow on charcoal-buffered yeast extract L. pneumophila causes Legionnaires disease
Methylomonas Gram-negative bacillus Use methane as source of carbon and energy
Proteus Gram-negative bacillus (pleomorphic) Common inhabitants of the human gastrointestinal tract motile produce urease opportunistic pathogens may cause urinary tract infections and sepsis
Pseudomonas Gram-negative bacillus Aerobic versatile produce yellow and blue pigments, making them appear green in culture opportunistic, antibiotic-resistant pathogens may cause wound infections, hospital-acquired infections, and secondary infections in patients with cystic fibrosis
Serratia Gram-negative bacillus Motile may produce red pigment opportunistic pathogens responsible for a large number of hospital-acquired infections
Shigella Gram-negative bacillus Nonmotile dangerously pathogenic produce Shiga toxin, which can destroy cells of the gastrointestinal tract can cause dysentery
Vibrio Gram-negative, comma- or curved rod-shaped bacteria Inhabit seawater flagellated, motile may produce toxin that causes hypersecretion of water and electrolytes in the gastrointestinal tract some species may cause serious wound infections
Yersinia Gram-negative bacillus Carried by rodents human pathogens Y. pestis causes bubonic plague and pneumonic plague Y. enterocolitica can be a pathogen causing diarrhea in humans

List two families of Gammaproteobacteria.


9.2 Oxygen Requirements for Microbial Growth

Ask most people “What are the major requirements for life?” and the answers are likely to include water and oxygen. Few would argue about the need for water, but what about oxygen? Can there be life without oxygen?

The answer is that molecular oxygen (O2) is not always needed. The earliest signs of life are dated to a period when conditions on earth were highly reducing and free oxygen gas was essentially nonexistent. Only after cyanobacteria started releasing oxygen as a byproduct of photosynthesis and the capacity of iron in the oceans for taking up oxygen was exhausted did oxygen levels increase in the atmosphere. This event, often referred to as the Great Oxygenation Event or the Oxygen Revolution , caused a massive extinction. Most organisms could not survive the powerful oxidative properties of reactive oxygen species (ROS), highly unstable ions and molecules derived from partial reduction of oxygen that can damage virtually any macromolecule or structure with which they come in contact. Singlet oxygen (O2•), superoxide ( O 2 − ) , ( O 2 − ) , peroxides (H2O2), hydroxyl radical (OH•), and hypochlorite ion (OCl − ), the active ingredient of household bleach, are all examples of ROS. The organisms that were able to detoxify reactive oxygen species harnessed the high electronegativity of oxygen to produce free energy for their metabolism and thrived in the new environment.

Oxygen Requirements of Microorganisms

Many ecosystems are still free of molecular oxygen. Some are found in extreme locations, such as deep in the ocean or in earth’s crust others are part of our everyday landscape, such as marshes, bogs, and sewers. Within the bodies of humans and other animals, regions with little or no oxygen provide an anaerobic environment for microorganisms. (Figure 9.19).

We can easily observe different requirements for molecular oxygen by growing bacteria in thioglycolate tube culture s. A test-tube culture starts with autoclaved thioglycolate medium containing a low percentage of agar to allow motile bacteria to move throughout the medium. Thioglycolate has strong reducing properties and autoclaving flushes out most of the oxygen. The tubes are inoculated with the bacterial cultures to be tested and incubated at an appropriate temperature. Over time, oxygen slowly diffuses throughout the thioglycolate tube culture from the top. Bacterial density increases in the area where oxygen concentration is best suited for the growth of that particular organism.

The growth of bacteria with varying oxygen requirements in thioglycolate tubes is illustrated in Figure 9.20. In tube A, all the growth is seen at the top of the tube. The bacteria are obligate (strict) aerobe s that cannot grow without an abundant supply of oxygen. Tube B looks like the opposite of tube A. Bacteria grow at the bottom of tube B. Those are obligate anaerobe s, which are killed by oxygen. Tube C shows heavy growth at the top of the tube and growth throughout the tube, a typical result with facultative anaerobe s. Facultative anaerobes are organisms that thrive in the presence of oxygen but also grow in its absence by relying on fermentation or anaerobic respiration, if there is a suitable electron acceptor other than oxygen and the organism is able to perform anaerobic respiration. The aerotolerant anaerobe s in tube D are indifferent to the presence of oxygen. They do not use oxygen because they usually have a fermentative metabolism, but they are not harmed by the presence of oxygen as obligate anaerobes are. Tube E on the right shows a “Goldilocks” culture. The oxygen level has to be just right for growth, not too much and not too little. These microaerophile s are bacteria that require a minimum level of oxygen for growth, about 1%–10%, well below the 21% found in the atmosphere.

Examples of obligate aerobes are Mycobacterium tuberculosis , the causative agent of tuberculosis and Micrococcus luteus , a gram-positive bacterium that colonizes the skin. Neisseria meningitidis , the causative agent of severe bacterial meningitis , and N. gonorrhoeae, the causative agent of sexually transmitted gonorrhea , are also obligate aerobes.

Many obligate anaerobes are found in the environment where anaerobic conditions exist, such as in deep sediments of soil, still waters, and at the bottom of the deep ocean where there is no photosynthetic life. Anaerobic conditions also exist naturally in the intestinal tract of animals. Obligate anaerobes, mainly Bacteroidetes , represent a large fraction of the microbes in the human gut. Transient anaerobic conditions exist when tissues are not supplied with blood circulation they die and become an ideal breeding ground for obligate anaerobes. Another type of obligate anaerobe encountered in the human body is the gram-positive, rod-shaped Clostridium spp. Their ability to form endospores allows them to survive in the presence of oxygen. One of the major causes of health-acquired infections is C. difficile, known as C. diff. Prolonged use of antibiotics for other infections increases the probability of a patient developing a secondary C. difficile infection. Antibiotic treatment disrupts the balance of microorganisms in the intestine and allows the colonization of the gut by C. difficile, causing a significant inflammation of the colon.

Other clostridia responsible for serious infections include C. tetani, the agent of tetanus, and C. perfringens, which causes gas gangrene . In both cases, the infection starts in necrotic tissue (dead tissue that is not supplied with oxygen by blood circulation). This is the reason that deep puncture wounds are associated with tetanus. When tissue death is accompanied by lack of circulation, gangrene is always a danger.

The study of obligate anaerobes requires special equipment. Obligate anaerobic bacteria must be grown under conditions devoid of oxygen. The most common approach is culture in an anaerobic jar (Figure 9.21). Anaerobic jars include chemical packs that remove oxygen and release carbon dioxide (CO2). An anaerobic chamber is an enclosed box from which all oxygen is removed. Gloves sealed to openings in the box allow handling of the cultures without exposing the culture to air (Figure 9.21).

Staphylococci and Enterobacteriaceae are examples of facultative anaerobes. Staphylococci are found on the skin and upper respiratory tract. Enterobacteriaceae are found primarily in the gut and upper respiratory tract but can sometimes spread to the urinary tract, where they are capable of causing infections. It is not unusual to see mixed bacterial infections in which the facultative anaerobes use up the oxygen, creating an environment for the obligate anaerobes to flourish.

Examples of aerotolerant anaerobes include lactobacilli and streptococci, both found in the oral microbiota. Campylobacter jejuni , which causes gastrointestinal infections, is an example of a microaerophile and is grown under low-oxygen conditions.

The optimum oxygen concentration , as the name implies, is the ideal concentration of oxygen for a particular microorganism. The lowest concentration of oxygen that allows growth is called the minimum permissive oxygen concentration . The highest tolerated concentration of oxygen is the maximum permissive oxygen concentration . The organism will not grow outside the range of oxygen levels found between the minimum and maximum permissive oxygen concentrations.

Check Your Understanding

  • Would you expect the oldest bacterial lineages to be aerobic or anaerobic?
  • Which bacteria grow at the top of a thioglycolate tube, and which grow at the bottom of the tube?

Case in Point

An Unwelcome Anaerobe

Charles is a retired bus driver who developed type 2 diabetes over 10 years ago. Since his retirement, his lifestyle has become very sedentary and he has put on a substantial amount of weight. Although he has felt tingling and numbness in his left foot for a while, he has not been worried because he thought his foot was simply “falling asleep.” Recently, a scratch on his foot does not seem to be healing and is becoming increasingly ugly. Because the sore did not bother him much, Charles figured it could not be serious until his daughter noticed a purplish discoloration spreading on the skin and oozing (Figure 9.22). When he was finally seen by his physician, Charles was rushed to the operating room. His open sore, or ulcer, is the result of a diabetic foot .

The concern here is that gas gangrene may have taken hold in the dead tissue. The most likely agent of gas gangrene is Clostridium perfringens , an endospore-forming, gram-positive bacterium. It is an obligate anaerobe that grows in tissue devoid of oxygen. Since dead tissue is no longer supplied with oxygen by the circulatory system, the dead tissue provides pockets of ideal environment for the growth of C. perfringens.

A surgeon examines the ulcer and radiographs of Charles’s foot and determines that the bone is not yet infected. The wound will have to be surgically debrided (debridement refers to the removal of dead and infected tissue) and a sample sent for microbiological lab analysis, but Charles will not have to have his foot amputated. Many diabetic patients are not so lucky. In 2008, nearly 70,000 diabetic patients in the United States lost a foot or limb to amputation, according to statistics from the Centers for Disease Control and Prevention. 1

Detoxification of Reactive Oxygen Species

Aerobic respiration constantly generates reactive oxygen species (ROS), byproducts that must be detoxified. Even organisms that do not use aerobic respiration need some way to break down some of the ROS that may form from atmospheric oxygen. Three main enzymes break down those toxic byproducts: superoxide dismutase, peroxidase, and catalase. Each one catalyzes a different reaction. Reactions of type seen in Reaction 1 are catalyzed by peroxidase s.

In these reactions, an electron donor (reduced compound e.g., reduced nicotinamide adenine dinucleotide [NADH]) oxidizes hydrogen peroxide , or other peroxides, to water. The enzymes play an important role by limiting the damage caused by peroxidation of membrane lipids. Reaction 2 is mediated by the enzyme superoxide dismutase (SOD) and breaks down the powerful superoxide anions generated by aerobic metabolism:

The enzyme catalase converts hydrogen peroxide to water and oxygen as shown in Reaction 3.

Obligate anaerobes usually lack all three enzymes. Aerotolerant anaerobes do have SOD but no catalase. Reaction 3, shown occurring in Figure 9.23, is the basis of a useful and rapid test to distinguish streptococci, which are aerotolerant and do not possess catalase, from staphylococci, which are facultative anaerobes. A sample of culture rapidly mixed in a drop of 3% hydrogen peroxide will release bubbles if the culture is catalase positive.

Bacteria that grow best in a higher concentration of CO2 and a lower concentration of oxygen than present in the atmosphere are called capnophiles . One common approach to grow capnophiles is to use a candle jar . A candle jar consists of a jar with a tight-fitting lid that can accommodate the cultures and a candle. After the cultures are added to the jar, the candle is lit and the lid closed. As the candle burns, it consumes most of the oxygen present and releases CO2.

Check Your Understanding

  • What substance is added to a sample to detect catalase?
  • What is the function of the candle in a candle jar?

Clinical Focus

Part 2

The health-care provider who saw Jeni was concerned primarily because of her pregnancy. Her condition enhances the risk for infections and makes her more vulnerable to those infections. The immune system is downregulated during pregnancy, and pathogens that cross the placenta can be very dangerous for the fetus. A note on the provider’s order to the microbiology lab mentions a suspicion of infection by Listeria monocytogenes , based on the signs and symptoms exhibited by the patient.

Jeni’s blood samples are streaked directly on sheep blood agar , a medium containing tryptic soy agar enriched with 5% sheep blood. (Blood is considered sterile therefore, competing microorganisms are not expected in the medium.) The inoculated plates are incubated at 37 °C for 24 to 48 hours. Small grayish colonies surrounded by a clear zone emerge. Such colonies are typical of Listeria and other pathogens such as streptococci the clear zone surrounding the colonies indicates complete lysis of blood in the medium, referred to as beta-hemolysis (Figure 9.24). When tested for the presence of catalase, the colonies give a positive response, eliminating Streptococcus as a possible cause. Furthermore, a Gram stain shows short gram-positive bacilli. Cells from a broth culture grown at room temperature displayed the tumbling motility characteristic of Listeria (Figure 9.24). All of these clues lead the lab to positively confirm the presence of Listeria in Jeni’s blood samples.

Jump to the next Clinical Focus box. Go back to the previous Clinical Focus box.


An Example of Phylum Platyhelminthes: Echinococcus

Echinococcus Granulosa lives in the small intes­tine of dogs and allied animals. The dog is the optimum definitive host. The larval stage is passed in sheep, cattle, pig or man which represent the intermediate hosts of the para­site. These animals, therefore, serve as the common reservoirs of the hydatid diseases.

Structure of Echinococcus Granulosa:

This parasite is the smallest tapeworm (in adult stage) of medical importance.

Echinococcus Granulosa measure 5-8 mm in length and are usually with three segments plus a scolex and a neck (Fig. 14.22). It has a typical taenioid scolex with four deep, well-developed suckers and an anteriorly located retractile rostellum beset with 30-36 hooklets. The scolex contains internal con­centration of nervous and excretory systems.

Behind the scolex lies the un-segmented proliferous neck region. The first proglottid has no definite organisation but the begin­ning of genital organs is marked by the presence of germinal mass. The second proglottid is mature and contains fully de­veloped genital organs. The last proglottid is gravid and relatively larger than other two segments. It contains only the uterus filled with eggs.

The eggs of Echinococcus Granulosa are ovoid in shape. The egg contains hexacanth embryo with 3 pairs of hooks.

Life Cycle of Echinococcus Granulosa:

The eggs produced by Echinococcus Granulosa in the intestine of dog or any other suitable primary host pass out along with the faeces and are ingested by the intermediate host with contaminated food or drink. The eggs, when swallowed, pass down the oesophagus into the stomach. Inside the stomach the shell wall is digested and the active hexacanth embryos (onchospheres) hatch out in the duodenum.

The liberated onchospheres bore their ways through the intestinal wall and enter the radicles of the portal vein. Whenever the onchospheretles, it forms a hydratid cyst. The young larva transforms into a hollow bladder. The cyst is double-walled. The outer layer is laminated while the inner one is germinative. Fig. 14.23 shows the scheme of a hy­datid cyst formation.

The young larva of Echinococcus Granulosa changes into a hollow bladder around which the host adds an enveloping fibrous cyst wall. With the advent of maturity, the inner surface begins to produce hollow brood cap­sules.

The brood capsule is formed by the proliferation of the cells from the germinative layer about eight months after the begin­ning of the cyst formation. It starts as small nuclear mass which grows and becomes vacuolated to form a small vesicle.

The brood capsules remain attached by slender stalks and often set free into the fluid-filled cavity of the mother cyst. As the cyst grows larger, more brood capsules develop.

The older brood capsules begin to differentiate a number of scolices on their inner walls. The mother cysts may, as a result of intracystic pressure, develop hernia-like buds which detach themselves and continue their devel­opment independently as daughter cyst.

Formation of Daughter Cysts:

The mother hydatid cyst develops daughter cysts. The daughter cysts are generally endogenous in origin while the exogenous daughter cysts occur rarely. The endogenous cysts arise from the germinative layer. The daughter cyst is almost identical to the mother cyst. Each scolex developed from the daughter cyst has the property of becoming a new hydatid in an intermediate host.

Transmission of Disease by Echinococcus Granulosa:

The end product of the hydatid cyst is the production of the scolices and each scolex has the power to develop into the strobilate worm, when ingested by any primary host (especially dog). As a sin­gle hydatid may contain thousands of brood capsules and each brood capsule may give origin to a large number of scolices, the number of strobilate worms resulting from such ingestion of a single hydated cyst be­come numerous.

Attainment of strobilate stage in the intestine of dog requires 3-10 weeks after ingestion. Man becomes infected in a variety of ways—viz., by swallowing food or drinks contaminated with infested canine faeces or by handling the infested dogs. The life cycle of Echinococcus granulosa is schematically represented in (Fig. 14.24).

Other Cestodes of Echinococcus Granulosa:

Over 1,500 species of cestodes are known to live as parasites on different animals rang­ing from fish to mammals (Fig. 14.25). Pri­mary host harbours the adult stages while embryonic stages occur in secondary hosts. Infection is caused when the secondary hosts are eaten by the primary hosts.

Hymenolepis nana:

Intermediate host is usually absent. Adults occur in the lumen of the intestine of man, and the larvae live in the intestinal villi. Proglottids numbering about 200 are popu­larly known as dwarf tapeworm. It meas­ures about 10-45 mm.

Taenia saginata:

Adult infects man, larvae live in cattle, proglottids are 2,000 in number (Fig. 14.25). T. saginata is a more common cestode parasite in man, because it uses cattle as its intermediate host. It resembles T. sobium but the rostellum is without hooks and the mode of branching of gravid uterus is different.

Taenia pisiformis:

Adults occur in the dog or cat, larvae stay in the liver and mesenteries of rabbit.

Moniezia expansa:

Adults reside in sheep, larvae remain in mites. It extends up to six metres in length (Fig. 14.25).

Diphyllobothrium latum:

Diphyllobothrium latum (also known as Dibothriocephalus latum), commonly called the fish-tapeworm, is the largest known cestode measuring about 18 metres in length. The number of proglottids varies from 3000- 4000. It has an extremely elongated slender neck.

The scolex has two slit-like bothria (Fig. 14.25) but hooks are absent. The sec­ondary hosts of this form are crustaceans (first host) and various freshwater fishes are the second host. Man or other carnivorous animals are its primary hosts.

Adaptive Features in Cestodes:

(A) The covering of the cestodes is not digestable by the host’s digestive juices and it is permeable to water,

(B) The osmotic pressure inside the body of the parasite is lower than the sur­rounding medium,

(D) Glycogen and lipid contents in the body tissues of cestodes are high and protein content is less,

(E) In the absence of oxygen it can re­spire anaerobically,

(F) The bladder of cysticercus is digested by the digestive juice but the scolex escapes digestion,