Why do anaerobic bacteria require a low redox potential for their culture media?

Why do anaerobic bacteria require a low redox potential for their culture media?

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I'm reading Clostridium ,which is an anaerobic bacteria in my microbiology text and it says,

of more importance than the absence of oxygen is the provision of a sufficiently low redox potential in the medium. Third can be achieved by adding unsaturated fatty acids, ascorbic acid, glutathione, cysteine, etc.

Why? Do they reduce oxygen free radicals or something? Well, are these present in Clostridium's natural habitat?

The higher the redox potential is, the higher the oxygen dissolved in the media will be. Lowering this potential insure that the oxygen concentration is lower and that the anaerobe will be able to grow easily.

Several steps can be done while preparing the media such as boiling to remove oxygen and adding chemical which reduces this redox potential to dissolve oxygen back into the medium.

As for the natural habitat, they naturally have no or really really low amounts of oxygen. If the habitats containing little oxygen have other molecules able to reduce redox potential is a good question.

Lowering the redox potential is mainly important for the medium since there is oxygen involved (hard to remove 100% while preparing media).

I hope my answer helps.


Culture media contains nutrients and physical growth parameters necessary for microbial growth. All microorganisms cannot grow in a single culture medium and in fact many can’t grow in any known culture medium. Organisms that cannot grow in artificial culture medium are known as obligate parasites. Mycobacterium leprae, rickettsias, Chlamydias, and Treponema pallidumareobligate parasites. Bacterial culture media can be distinguished based on composition, consistency, and purpose.


  1. Solid medium: – Solid medium contains agar at a concentration of 1.5-2.0% or some other, mostly inert solidifying agent. Solid medium has physical structure and allows bacteria to grow in physically informative or useful ways (e.g. as colonies or in streaks). Solid medium is useful for isolating bacteria or for determining the colony characteristics of the isolate.
  2. Semisolid media: – They are prepared with agar at concentrations of 0.5% or less. They have soft custard like consistency and are useful for the cultivation of microaerophilic bacteria or for determination of bacterial motility.
  3. Liquid (Broth) medium: – These media contain specific amounts of nutrients but don’t have trace of gelling agents such as gelatine or agar. Broth medium serves various purposes such as propagation of large number of organisms, fermentation studies, and various other tests. e.g. sugar fermentation tests, MR-VR broth.


  1. Synthetic or chemically defined medium: – A chemically defined medium is one prepared from purified ingredients and therefore whose exact position is known.
  2. Non-synthetic or chemically undefined medium: – Non-synthetic medium contains at least one component that is neither purified nor completely characterised nor even completely consistent from batch to batch. Often these are partially digested proteins from various organism sources. Nutrient broth, for example, is derived from cultures of yeasts.


Many special purpose media are needed to facilitate recognition, enumeration, and isolation of certain types of bacteria. To meet these needs, numerous media are available.

  1. General purpose media/ Basic media: – Basal media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient broth and nutrient agar are considered as basal medium. These media are generally used for the primary isolation of microorganisms.
  2. Enriched medium (Added growth factors): – Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them enriched media. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Blood agar, chocolate agar, Loeffler’s serum slope etc are few of the enriched media. Blood agar is prepared by adding 5-10% (by volume) blood to a blood agar base. Chocolate agar is also known as heated blood agar or lysed blood agar.
  3. Selective and enrichment media: – They are designed to inhibit unwanted commensal or contaminating bacteria and help to recover pathogen from a mixture of bacteria. While selective media are agar based, enrichment media are liquid in consistency. Both these media serve the same purpose. Any agar media can be made selective by addition of certain inhibitory agents that don’t affect the pathogen of interest. Various approaches to make a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these. –Selective medium: –Differential growth suppression. Selective medium is designed to suppress the growth of some microorganisms while allowing the growth of others. Selective medium is agar based (solid) medium so that individual colonies may be isolated.

Examples of selective media include:

  • Thayer Martin Agar used to recover N. gonorrhoeae contains antibiotics vancomycin, colistin and nystatin.
  • Mannitol Salt Agar and Salt Milk Agar used to recover S. aureus contains 10% NaCl.
  • Potassium tellurite medium used to recover C. diphtheriae contains 0.04% potassium tellurite.
  • MacConkey’s Agar used for Enterobacteriaceae members contains bile salt that inhibits most gram-positive bacteria.
  • Pseudogel Agar (Cetrimide Agar) used to recover P. aeruginosa contains cetrimide (antiseptic agent).
  • Crystal Violet Blood Agar used to recover S. pyogenes contains 0.0002% crystal violet.
  • Lowenstein Jensen Medium used to recover M. tuberculosis is made selective by incorporating malachite green.
  • Wilson and Blair’s Agar for recovering S. typhi is rendered selective by the addition of dye brilliant green.
  • Selective media such as TCBS Agar used for isolating V. cholerae from faecal specimens have elevated pH (8.5-8.6), which inhibits most other bacteria. –Enrichment culture medium: –Enrichment medium is used to increase the relative concentration of certain microorganisms in the culture prior to plating on solid selective medium. Unlike selective media, enrichment culture is typically used as broth medium. Enrichment media are liquid media that also serves to inhibit commensals in the clinical specimen. Selenite F broth, tetrathionate broth and alkaline peptone water (APW) are used to recover pathogens from faecal specimens.
  1. Differential/ indicator medium: – Certain media are designed in such a way that different bacteria can be recognised on the basis of their colony colour. Various approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that utilise them appear as differently coloured colonies. Such media are called differential media or indicator media. Differential media allow the growth of more than one microorganism of interest but with morphologically distinguishable colonies.

Examples of differential media include:

  • Mannitol salts agar (mannitol fermentation = yellow)
  • Blood agar (various kinds of haemolysis i.e. α, β and γ haemolysis)
  • Mac Conkey agar (lactose fermenters, pink colonies whereas non- lactose fermenter produces pale or colourless colonies.
  • TCBS (Vibrio cholerae produces yellow colonies due to fermentation of sucrose)
  1. Transport media: – Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms or commensals. This can be achieved by using transport media. Such media prevent drying (desiccation) of specimen, maintain the pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria. Some of these media (Stuart’s & Amie’s) are semi-solid in consistency. Addition of charcoal serves to neutralize inhibitory factors.

* Cary Blair transport medium and Venkatraman Ramakrishnan (VR) medium are used to transport faeces from suspected cholera patients.

* Sach’s buffered glycerol saline is used to transport faeces from patients suspected to be suffering from bacillary dysentery.

* Pike’s medium is used to transport streptococci from throat specimens.

  1. Anaerobic media: Anaerobic bacteria need special media for growth because they need low oxygen content, reduced oxidation –reduction potential and extra nutrients.

Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K. Such media may also have to be reduced by physical or chemical means. Boiling the medium serves to expel any dissolved oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can render a medium reduced. Before use the medium must be boiled in water bath to expel any dissolved oxygen and then sealed with sterile liquid paraffin.

Robertson Cooked Meat (RCM) medium that is commonly used to grow Clostridium species contains a 2.5 cm column of bullock heart meat and 15 ml of nutrient broth. Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast extract and casein hydrolysate.

Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in the medium. Under reduced condition, methylene blue is colourless.


Iron (Fe) has long been a recognized physiological requirement for life, yet for many microorganisms that persist in water, soils and sediments, its role extends well beyond that of a nutritional necessity. Fe( II ) can function as an electron source for iron-oxidizing microorganisms under both oxic and anoxic conditions and Fe( III ) can function as a terminal electron acceptor under anoxic conditions for iron-reducing microorganisms. Given that iron is the fourth most abundant element in the Earth's crust, iron redox reactions have the potential to support substantial microbial populations in soil and sedimentary environments. As such, biological iron apportionment has been described as one of the most ancient forms of microbial metabolism on Earth, and as a conceivable extraterrestrial metabolism on other iron-mineral-rich planets such as Mars. Furthermore, the metabolic versatility of the microorganisms involved in these reactions has resulted in the development of biotechnological applications to remediate contaminated environments and harvest energy.

Chapter 22: Anaerobes of Clinical Importance

-Gram (+)
-Pathogenic bacterium of the genus Clostridium
-Everpresent in nature and can be found as a normal component of decaying vegetation, marine sediment, the intestinal tract of humans and other vertebrates, insects, and soil.

-Cycloserine-cefoxitin-fructose agar (CCFA)
(Selective and differential for C. difficile--> Yellow "ground glass" colonies
Horse stable odor)

-Lecithinase and lipase reactions

-Spot indole, rapid urease, gelatin hydrolysis

A condition of necrotic tissue damage, specific to muscle tissue. Bacteria cause myonecrosis by specific exotoxins (alpha toxin, lethal toxin, and hemolytic toxin)

-Acute onset of severe abdominal pain and diarrhea, which is often bloody, and may be accompanied by vomiting

-Followed by necrotic inflammation of the small intestines, at times leading to bowel perforation

-Gram (+)
-Pathogenic bacteria of the genus Clostridium
-Spore-forming, heavily swarming

Antarctic strict anaerobic microbiota from Deschampsia antarctica vascular plants rhizosphere reveals high ecology and biotechnology relevance

The Antarctic soil microbial community has a crucial role in the growth and stabilization of higher organisms, such as vascular plants. Analysis of the soil microbiota composition in that extreme environmental condition is crucial to understand the ecological importance and biotechnological potential. We evaluated the efficiency of isolation and abundance of strict anaerobes in the vascular plant Deschampsia antarctica rhizosphere collected in the Antarctic’s Admiralty Bay and associated biodiversity to metabolic perspective and enzymatic activity. Using anaerobic cultivation methods, we identified and isolated a range of microbial taxa whose abundance was associated with Plant Growth-Promoting Bacteria (PGPB) and presences were exclusively endemic to the Antarctic continent. Firmicutes was the most abundant phylum (73 %), with the genus Clostridium found as the most isolated taxa. Here, we describe two soil treatments (oxygen gradient and heat shock) and 27 physicochemical culture conditions were able to increase the diversity of anaerobic bacteria isolates. Heat shock treatment allowed to isolate a high percentage of new species (63.63 %), as well as isolation of species with high enzymatic activity (80.77 %), which would have potential industry application. Our findings contribute to the understanding of the role of anaerobic microbes regarding ecology, evolutionary, and biotechnological features essential to the Antarctic ecosystem.

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Candida albicans causes 80% to 90% of vaginal fungal infections, and other Candida species and Torulopsis cause the remainder. 34 These saprophytic fungi can be isolated in small numbers from 5% to 20% of asymptomatic women. Symptoms usually only result when these organisms proliferate to large numbers.

The true incidence of vulvovaginal candidiasis is unknown. Women who carry C. albicans in the genital tract can be asymptomatic or have symptoms from severe inflammation. Symptoms reflect the host immune response. Diagnosis without the benefit of microscopy or culture indicates that one half of women diagnosed with candidiasis instead have other conditions. 1 By college age, about one half of women have one physician-diagnosed episode of candidiasis. 35 Candidiasis increases after menarche, in part related to initiation of sexual activity. 36 , 37 The rate of candidiasis is increased during pregnancy. Postmenopausal women given estrogen replacement can have candidiasis.

The frequency of intercourse is related to candidiasis. 36 , 37 The number of sexual partners 36 and oral-genital contact 37 do not increase the rate of candidiasis. Candidiasis appears weakly related to high-dose estrogen contraceptive pills. 38 Sexual intercourse with the use of nonoxynol-9 spermicide appears to increase colonization with Candida. 39 Douching with commercial products appears to temporarily alter vaginal flora 32 and intermittently has been associated with recurrent Candida infection. 36 , 37

Antibiotics have been strongly related to candidiasis, 40 and this association may be especially important for women with recurrent infection. 41 It is not clear whether antibiotics kill bacteria such as Lactobacillus that may inhibit the growth of Candida or use other mechanisms. 6

Diet may play little in the role of candidiasis. 42 Hygiene practices, including frequent bowel movements, wipe direction after bowel movement, type of menstrual protection, underclothing fabric, and tight clothing appear to play no role in candidiasis. 37 Immunosuppression from HIV infection is associated with C. torulopsis infection 34 and increased rates of esophageal and perhaps vaginal C. albicans infection. Uncontrolled diabetes is associated with candidiasis, particularly with unresponsive infections.

Recurrence of typical severe symptoms such as pruritus and vulvar irritation often represent candidiasis, but atypical or minimal symptoms are incorrectly self-diagnosed in about one half of cases. 43 Besides the finding of a low pH and the microscopic findings previously discussed, cultures are necessary for women with suspected candidiasis for whom the microscopic results for Candida are negative. Among culture-positive women with vulvar symptoms of external dysuria, pruritus, swelling, or redness, the KOH wet mount was positive for only about 60%, leaving 30% of women undiagnosed by KOH. 4

Uncomplicated candidiasis refers to sporadic infrequent episodes with mild to moderate symptoms in a normal, nonpregnant woman. Virtually all uncomplicated infections are caused by C. albicans. Complicated candidiasis refers to recurrent infection, infection with severe symptoms or infection in women who are pregnant, diabetic, or immunosuppressed. Many cases of complicated infection are caused by non-albicans species.


Topical azole therapy remains the first choice to treat infrequent acute candidiasis. Azoles are fungistatic by their inhibition of ergosterol (and membrane) synthesis. Candida organisms are killed by the host lymphocytes through cell-mediated immune mechanisms. Only a limited fungicidal effect can be achieved by a high concentration of azoles that produce direct membrane damage. Topical azoles are effective, well tolerated, and relatively inexpensive. The products available include buconazole (Femstat), clotrimazole (Gyne-Lotrimin, Mycelex), miconazole (Monistat), and terconazole (Terazol). A wide range of doses in cream and suppository forms are available (Table 2). For some preparations, the treatment interval has been increased to twice daily or the dose has been increased from 100 to 200 mg while the length of medication was reduced concurrently from 7 to 3 days. Cure rates for the 3-day course have been equal to longer courses for uncomplicated candidiasis. Short-term (7- to 30-day) cure rates with topical azoles used for 3 to 7 days are usually 80% to more than 90%. There is no suggestion that the cure rates differ between various different azoles or between the suppository and the cream form.

TABLE 2. Recommended Azole Regimens for the Treatment of Vulvovaginal Candidiasis

Clotrimazole (Gyne-Lotrimin, Mycelex)

I do not recommend single-dose therapy, because clinical experience suggests that 1-day courses are less effective than published reports indicate. This may be explained in part by the tendency to include women with mild symptoms of short duration and no prior candidiasis in the studies. Usually, only short-term (7- to 30-day) clinical cure rates are reported, and higher mycologic failure rates occur at 30 days when patients are given a single dose compared with a longer course of therapy. 44 However, it is not certain that the increased rate of Candida recovery after treatment is related to increased rates of subsequent clinical candidiasis, and the duration of therapy may be of less importance in uncomplicated sporadic infection than in cases of complicated infection.

The oral azole fluconazole makes oral medication a safe choice for a vaginal candidiasis. A one-time 150-mg oral dose is effective for patients with mild to moderate symptoms. 45 This drug has become popular for patients because vaginal cream is not used and for physicians because short-term use is without liver toxicity, a problem that prevented widespread use of ketoconazole. However, the dose often needs to be repeated in 4 to 5 days for very symptomatic patients because of high failure rates that otherwise exceed failure rates of local therapy. 45 Physicians need to be aware of drug interaction between fluconazole and antihistamine drugs that prolong the QTc interval, oral hypoglycemics, Coumadin, and other drugs (consult a drug reference). 46 Among nonimmunosuppressed patients, widespread use probably does not lead to resistance.

Topical polyene therapy consisting of nystatin has been largely replaced by topical azole treatment. Nystatin is well tolerated and inexpensive, but cure rates of 50% to 80% are lower than those for azoles. Nystatin is a second-line choice in treating uncomplicated candidiasis.

Boric acid capsules (600 mg or boric acid powder in a 0 size gelatin capsules) inserted twice daily for 14 days provides clinical cure rates similar to the topical azoles. 47 The boron ion has not been detected in blood, 48 and boric acid is inexpensive and well tolerated. However, boric acid can produce esophageal ulcers if inadvertently swallowed, and the preparation should be stored in bottles with childproof caps, kept in a locked medicine cabinet, and used with caution when small children are in the household. Because of unproven fetal safety, boric acid should not be used in pregnancy. Gentian violet treatment is effective for the treatment of candidiasis, but the staining of clothing and skin limits its use to the rare case unresponsive to other medication. Potassium sorbate and povidone iodine have limited effectiveness against candidiasis.

Local Candida therapy is usually well tolerated, and reactions are unusual. If increased vaginal irritation occurs with use, the medication should immediately stop and changed to a different preparation. Most of these irritations result from reactions to &ldquoinactive&rdquo compounds in the cream vehicle.


Patients with return of symptoms a few days after completing a course of medication usually have taken only a short course or been noncompliant. Many times, a longer course or different preparation suffices. Resistance of Candida to antifungal medication is uncommon, and some patients with continual symptoms have other diseases. Patients with a rapid recurrence need to have candidiasis documented by a KOH wet mount, culture, or both. Patients without objective evidence of Candida but with persistent symptoms should be scrutinized for vaginitis or vulvitis caused by other conditions. Neurodermatitis, lichen planus, lichen sclerosis, burning vulvar syndrome, and minor vestibular gland inflammation should be considered.

The small number of patients with drug resistance usually has a reduction of symptoms while on therapy but a rapid recurrence of symptoms after medication is stopped. C. albicans is almost uniformly sensitive to all azoles. However, resistance to azoles is more common among unusual fungi such as Candida glabrata, Candida tropicalis, or other non-albicans Candida species, 49 and possible resistance should be considered if these fungi are present. Terconazole treatment may be tried because it has greater activity than other azoles against C. glabrata and C. tropicalis. Patients can be switched to the nonazole drugs, nystatin or boric acid. Boric acid is more effective than fluconazole for treatment of non-albicans vulvovaginal candidiasis. 49 , 50 Gentian violet therapy may also be of benefit. In rare instances, none of the topical medications is effective in eliminating vaginal fungi, and nystatin or boric acid need to be given every 2 to 3 days indefinitely to suppress rather than eliminate resistant fungi.

Treatment failures have been so infrequent for candidiasis that systematic studies have not been performed to determine the causes of treatment failure. The follow-up period has been too short (30 days or less) to accurately assess the frequency of failures. Compliance failure, pseudofailure due to a reaction to vehicle, and drug resistance have been mentioned. Two other prominent theories of treatment failure require discussion.

The first theory is that recurrent vaginal candidiasis results from gastrointestinal tract Candida. Candida organisms are found in higher rates in the oral cavity and gastrointestinal tract of patients with candidiasis than controls. 51 However, most patients carry Candida in the gastrointestinal tract without developing vaginal candidiasis, and in longitudinal studies, vaginal candidiasis has not been related to gastrointestinal Candida. Oral nystatin therapy has provided mixed results. Oral nystatin has not had a dramatic effect, although a slight statistically significant reduction in subsequent Candida vulvovaginitis occurred in the group receiving oral nystatin compared with placebo. 52 Eating 4 ounces of lactobacilli-containing yogurt twice daily has also provided a modest reduction in recurrence. 53

The second theory involves sexual transmission of recurrent candidiasis. Male sexual partners of patients with vaginal candidiasis have higher rates of genital colonization than controls. 54 Sexual transmission probably does occur for the small number (estimated to be 10% to 15%) of males who have Candida balanitis concurrent with their partner's vaginal candidiasis. However, only 20% of male sexual partners are colonized with Candida, and treatment of males has not decreased vaginal candidiasis for the female. Most males appear passively colonized by the female rather than play a major role in recurrent vaginal candidiasis.

Temporary reduction of vulvar symptoms occurs with a sitz bath followed by superdrying of the skin with a hairdryer using the low heat setting. Topical azole creams can also be prescribed for direct vulvar use, but little information is available on its effectiveness.

A diet to reduce high carbohydrate levels has been of some benefit for a subset of patients who developed candidiasis shortly after ingesting an unusually high amount of sugar. Among diabetics, candidiasis treatment is usually not successful until glucose levels are controlled. The avoidance of foods made from yeast has been popularized, but scientific documentation of effectiveness is lacking. Yeast organisms in food are not the same species that cause vaginitis. The yeast that cause vaginitis are so prevalent in the environment and on the skin that is difficult to believe that a yeast-free diet influences vaginal candidiasis. Cessation of antibiotic therapy can be considered, when possible, in a small number of patients on chronic antimicrobials. Cessation of oral contraceptive therapy is controversial and probably of limited benefit. Abandonment of tight, poorly ventilated clothing is probably of little benefit.


A significant subset of patients have been identified with chronic or frequently recurrent (at least four annual) episodes of vaginal candidiasis. These patients accumulate to represent a significant number of patients with vaginal candidiasis attending certain clinics.

It is not clear whether patients with chronic recurrent candidiasis have a limited immunologic defect in which lymphocytes do not kill Candida organisms in the vagina or other reasons for recurrence. Recurrence is not related to unusual or drug-resistant strains of Candida, and few patients are diabetic or taking oral contraceptives, immunosuppressive drugs, or antibiotics. Women with frequent recurrences of vaginal candidiasis or candidiasis that do not respond to treatment should be considered for HIV testing. However, few women with recurrent candidiasis have HIV or any other commonly recognized factor that predisposes patients to candidiasis.

Symptomatic disease often is difficult to manage, but an important breakthrough was chronic suppressive therapy similar to that used for urinary tract infection. The initial study used a therapeutic dose of 400 mg of oral ketoconazole for 14 days, followed by a maintenance dose for 6 months. 55 Potential liver toxicity and expense limits ketoconazole use, and the 14-day regimen now should consist of fluconazole given every 4 days or vaginal azole. In a controlled study, ketoconazole produced a recurrence rate at 6 months of 70% for those on placebo and 5% for those on daily ketoconazole. 55 Practical maintenance regimens include biweekly or weekly topical azole administration. 56 Similar results are achieved using oral itraconazole or fluconazole as maintenance or topical boric acid. 57 Recurrence of vaginal candidiasis may resume at the same rates as before suppression when maintenance therapy ceases. 55

Systemic absorption of azoles or nystatin from the vagina is so limited that both can be safely used during all trimesters of pregnancy. Boric acid, fluconazole, itraconazole, and ketoconazole should not be used during pregnancy. Compared with nonpregnant women, candidiasis in pregnancy is more resistant to treatment, is more likely to relapse, and responds better to 7- or even 14-day therapy compared with 1- to 3-day therapy.

Author Contributions

SO did the literature review, writing of the initial draft and revisions, figure design, and project administration. BB contributed to literature review, revisions and provided critical review. DA, JR, PL, and OL provided critical review and commentaries. JJ and LH had the rationale for this work, contributed to literature review, and supervised manuscript revisions. LH additionally contributed to figure design. JJ additionally contributed to photography of the blood culture bottles.


Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H2O2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H2O2. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality.

The increasing incidence of antibiotic-resistant infections coupled with a declining antibiotic pipeline has created a global public health threat (1 ⇓ ⇓ ⇓ ⇓ –6). Therefore there is a pressing need to expand our conceptual understanding of how antibiotics act and to use insights gained from such efforts to enhance our antibiotic arsenal. It has been proposed that different classes of bactericidal antibiotics, regardless of their drug–target interactions, generate varying levels of deleterious reactive oxygen species (ROS) that contribute to cell killing (7, 8). This unanticipated notion, built upon important prior work (9 ⇓ –11), has been extended and supported by multiple laboratories investigating wide-ranging drug classes (e.g., β-lactams, aminoglycosides, and fluoroquinolones) and bacterial species (e.g., Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baumannii, Burkholderia cepecia, Streptococcus pneumonia, Enterococcus faecalis) using independent lines of evidence (12 ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ –39). Importantly, these ongoing efforts have served to refine aspects of the initial model and show that antibiotic-induced ROS generation is a more complex process than originally suggested, likely involving additional means of production.

The redox stress component of antibiotic lethality is hypothesized to derive from alterations to multiple core aspects of cellular physiology and stress response activation. Specifically, this component includes alterations to central metabolism, cellular respiration, and iron metabolism initiated by drug-mediated disruptions of target-specific processes and resulting cellular damage (Fig. 1A). Important support for this hypothesis can be found in pathogenic clinical isolates whose drug tolerance involves mutations in oxidative stress response and defense genes and not exclusively in drug target mutagenesis (40 ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ –48).

Bactericidal antibiotics promote the generation of toxic reactive species. (A) Bactericidal antibiotics of different classes are capable of inducing cell death by interfering with their primary targets and corrupting target-specific processes, resulting in lethal cellular damage. Target-specific interactions trigger stress responses that induce redox-related physiological alterations resulting in the formation of toxic reactive species, including ROS, which further contribute to cellular damage and death. (B) Treatment of wild-type E. coli with ampicillin (Amp, 5 μg/mL), gentamicin (Gent, 5 μg/mL), or norfloxacin (Nor, 250 ng/mL) induces ROS, detectable by several chemically diverse fluorescent dyes with ranging specificity. One-way ANOVA was performed to determine statistical significance against the no-dye autofluorescence control. The dyes used were 5/6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (Carboxy-H2DCFDA) 5/6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) 4-amino-5-methylamino-2´,7´-diflurorescein diacetate (DAF-FM) 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) 3′-(p-hydroxyphenyl) fluorescein (HPF) OxyBURST Green (Oxyburst) and Peroxy-Fluor 2 (PF2). (C) Treatment of a quinolone-resistant strain (gyrA17) with norfloxacin does not produce detectable ROS. Data shown reflect mean ± SEM of three or more technical replicates. Where appropriate, statistical significance is shown and computed against the no-treatment control or no-dye control (*P ≤ 0.05 **P ≤ 0.01 ***P ≤ 0.001).

Recent critiques of this evolving model have misinterpreted an essential aspect of the hypothesis. Specifically, these recent studies (49 ⇓ –51) are predicated on the notion that ROS are the sole arbiters of antibiotic lethality, thereby implying that the model suggests that antibiotics do not kill by disrupting their well-established, target-specific processes. However, the evolving model is completely consistent with the literature indicating that bactericidal antibiotics are capable of inducing lethal cellular damage via interference with target-specific processes, ultimately resulting in cell death. Rather than refute this traditional view of antibiotic action, the hypothesis extends it by suggesting that an additional component of toxicity results from ROS, which are generated as a downstream physiological consequence of antibiotics interacting with their traditional targets. In this respect, reactive species are thought to contribute causatively to drug lethality.

However, an important gap exists in our general understanding of how bacteria respond physiologically to antibiotic–target interactions on a system-wide level, how these responses contribute to antibiotic killing, and how the extracellular environment protects or exacerbates the intracellular contributions to cell death. Here, we use a multidisciplinary set of biochemical, enzymatic, biophysical, and genetic assays to address these issues and expand our understanding of antibiotic-induced physiological responses and factors contributing to antibiotic lethality. Data from the present work indicate that antibiotic lethality is accompanied by ROS generation and that such reactive species causatively contribute to antibiotic lethality.


Christian Tennert: Conceptualization (lead) Formal analysis (equal) Methodology (equal) Project administration (equal) Supervision (equal) Writing-original draft (equal) Writing-review & editing (equal). Ann-Christin Reinmuth: Data curation (equal) Formal analysis (equal) Investigation (equal) Visualization (equal). Katharina Bremer: Data curation (equal) Investigation (equal). Ali Al-Ahmad: Conceptualization (equal) Funding acquisition (equal) Methodology (equal) Writing-review & editing (equal). Lamprini Karygianni: Formal analysis (equal) Visualization (equal) Writing-review & editing (equal). Elmar Hellwig: Resources (lead) Supervision (equal) Writing-review & editing (equal). Kristin Vach: Formal analysis (lead) Methodology (equal) Writing-review & editing (equal). Petra Ratka-Krüger: Project administration (equal) Supervision (equal) Writing-original draft (equal). Annette Wittmer: Data curation (equal) Investigation (lead) Writing-review & editing (equal). Johan Peter Woelber: Conceptualization (equal) Methodology (equal) Project administration (equal) Supervision (equal) Writing-review & editing (equal).

A heavy inoculum of test organism is incubated in a broth containing nitrate. The organisms capable of producing the nitrate reductase enzyme reduce the nitrate, present in the broth, to nitrite which may then be further reduced to nitric oxide, nitrous oxide, or nitrogen.

The nitrate reduction test is based on the detection of nitrite and its ability to form a red compound when it reacts with sulfanilic acid to form a complex (nitrite-sulfanilic acid) which then reacts with a α-naphthylamine to give a red precipitate (prontosil), which is a water-soluble azo dye.

However, only when nitrate is present in the medium, red color will be produced. If there’s no red color in the medium after you’ve added sulfanilic acid and α-naphthylamine means only that nitrite is not present in the medium.

There is two explanations for this observation.

  1. The nitrate may not have been reduced the strain is nitrate-negative.
  2. The nitrate may have been reduced to nitrite which has then been completely reduced to nitric oxide, nitrous oxide, or nitrogen which will not react with the reagents that react with nitrite the strain is nitrate-positive.

Thus, when nitrite is not detected, it is necessary to test if the organism has reduced nitrate beyond nitrite. This can be done indirectly by adding small amount of Zinc powder to the culture. Zinc powder catalyses the reduction of nitrate to nitrite. The development of the red colour on addition of Zinc indicates that nitrate was not reduced by the organism which suggests that the test organism is not capable of reducing nitrate. If no color change occurs after the addition of zinc, this indicates that the organism reduced nitrate to one of the other nitrogen compounds and thus is a nitrate reducer.

Note: A Durham tube is placed in the nitrogen broth in order to detect deterioration of the broth before inoculation, as evidenced by gas formation in the tube and to identify denitrification by organisms that produce gas by alternate pathways.