Rhizopus spp- An Overview

Rhizopus spp is a genus of common saprophytic fungi that belong to the family Mucoraceae and the order Mucorales. They are also known as pin or bread molds because of their appearance on bread and other food products.

Rhizopus spp are widely distributed in nature and can be found on a variety of organic substrates, such as soil, decaying fruits and vegetables, jellies, syrups, leather, tobacco, peanuts, animal feces, and compost. They can also grow in indoor environments, such as homes and businesses, where they may cause spoilage of food or damage to materials.

Rhizopus spp are able to thrive in different environmental conditions, such as warm and humid or cool and dry climates. They can tolerate a wide range of pH levels (from 2 to 11) and temperatures (from 5°C to 45°C). They can also grow in low oxygen or high carbon dioxide levels.

Rhizopus spp have both economic and medical importance. Some species are used in industrial processes, such as the production of lactic acid, fumaric acid, cortisone, biotin, alcoholic beverages, and fermented foods (e.g., tempeh). However, some species are also responsible for diseases in plants and animals, including humans. The most serious human infection caused by Rhizopus spp is mucormycosis (also known as zygomycosis), which is a rare but often fatal disease that affects the sinuses, lungs, brain, skin, and other organs. Mucormycosis mainly occurs in immunocompromised individuals or those with underlying conditions, such as diabetes mellitus, malnutrition, malignancies, renal failure, organ transplant, burns, or AIDS.

In this article, we will discuss the morphological and cultural characteristics of Rhizopus spp, the pathogenesis of Rhizopus spp in humans, animals, and plants, the clinical manifestations of mucormycosis/zygomycosis, the laboratory diagnosis of mucormycosis (Rhizopus spp), the treatment of mucormycosis, and some selected species of Rhizopus and their industrial uses.

Rhizopus spp are filamentous fungi that belong to the order Mucorales and the family Rhizopodaceae. They are cosmopolitan and ubiquitous, found on various organic substrates such as soil, decaying fruits and vegetables, bread, leather, and animal feces . They are also important pathogens of humans, animals, and plants, causing infections such as mucormycosis and fruit rot .

The morphology of Rhizopus spp is characterized by the following features:

• Mycelium: Rhizopus spp mycelium is a network of branching, thread-like hyphae. The hyphae are non-septate, which indicates that they lack cross-walls. This enables the hyphae to rapidly grow and spread .
• Hyphae: Rhizopus spp hyphae are of three types: stolons, rhizoids, and sporangiophores .
  • Stolons: These are horizontal hyphae that run along the surface of the substrate. They produce rhizoids and sporangiophores at regular intervals .
  • Rhizoids: These are short, root-like hyphae that grow downward from the stolons into the substrate. They anchor the fungus to the substrate and absorb nutrients .
  • Sporangiophores: These are vertical hyphae that grow upward from the stolons. They are usually unbranched and terminate in a spherical structure called a sporangium .
• Sporangia: These are round or oval structures that contain numerous spores for asexual reproduction. The spores are called sporangiospores and they are globose to ovoid, single-celled, hyaline to brown, and striate in many species . The sporangia are supported by a swollen base called a columella and a neck-like extension called an apophysis . After spore release, the columella and apophysis often collapse to form an umbrella-like structure.
• Zygospores: These are thick-walled spores that result from sexual reproduction. They are formed when two compatible and physiologically distinct mycelia fuse together. Zygospores germinate to produce new genetically different offspring .

Some of the cultural characteristics of Rhizopus spp include:

• Growth rate: Rhizopus spp grow rapidly on suitable media, with visible growth often observed within 24 to 48 hours .
• Colony morphology: The colonies of Rhizopus spp are fast-growing, cottony, and often fluffy in texture. They are initially white and turn grey or yellowish-brown with sporulation. The reverse pigmentation is white to pale .
• Temperature range: Rhizopus spp can grow over a wide range of temperatures, from 5°C to 45°C. However, some species have specific temperature preferences. For example, R. microsporus is thermotolerant and can grow up to 54°C, while R. variabilis is mesophilic and has a maximum growth temperature of 38°C.

Rhizopus spp are a group of fungi that can cause infections in humans, animals, and plants. The most common human infection caused by Rhizopus spp is mucormycosis, also known as zygomycosis. Plant diseases caused by Rhizopus spp are collectively known as rots, affecting various fruits and vegetables. Animal infections are less common, but can occur in livestock and pets.

Pathogenesis in human and animal hosts: Mucormycosis/Zygomycosis

Mucormycosis is a serious and often fatal infection that occurs mainly in immunocompromised individuals, such as those with diabetes mellitus, malignancies, organ transplantation, renal failure, burns, or AIDS . Additionally, patients with diabetic ketoacidosis or those receiving iron chelator deferoxamine are more susceptible to mucormycosis.

The infection is caused by inhalation or inoculation of spores of Rhizopus spp, which can germinate and invade the blood vessels and surrounding tissues of the host . The most common species involved are Rhizopus arrhizus (also known as R. oryzae), R. microsporus, and R. stolonifer .

Mucormycosis can manifest in different forms, depending on the site of infection and the extent of dissemination. The most common form is rhinocerebral mucormycosis, which affects the sinuses and the brain. Other forms include pulmonary mucormycosis, which affects the lungs; cutaneous mucormycosis, which affects the skin; gastrointestinal mucormycosis, which affects the digestive tract; and disseminated mucormycosis, which affects multiple organs.

The clinical manifestations of mucormycosis vary according to the form of infection, but generally include fever, pain, swelling, necrosis, ulceration, and tissue destruction. Some of the specific signs and symptoms of each form are:

• Rhinocerebral mucormycosis: sinusitis, nasal congestion, nasal discharge, sinus pain, facial swelling, erythema, necrosis of the palate or nasal septum, blurring of vision, ophthalmoplegia (paralysis of eye muscles), cranial neuropathies (nerve damage), lethargy, seizures, slurred speech, partial paralysis, brain abscess, altered consciousness, and coma.
• Pulmonary mucormycosis: cough, hemoptysis (coughing up blood), chest pain, dyspnea (difficulty breathing), pleural effusion (fluid accumulation in the chest cavity), pneumonia (lung inflammation), pulmonary infarction (tissue death due to lack of blood supply), pulmonary nodules (masses in the lungs), and respiratory failure.
• Cutaneous mucormycosis: skin inflammation, induration (hardening), erythema (redness), pain, swelling, blistering, ulceration, necrosis (tissue death), black eschar (scab), cellulitis (skin infection), and gangrene (tissue death due to infection).
• Gastrointestinal mucormycosis: abdominal pain, nausea, vomiting, hematemesis (vomiting blood), diarrhea, melena (black stools due to bleeding), bowel perforation (hole in the intestine), peritonitis (inflammation of the abdominal lining), bowel infarction (tissue death due to lack of blood supply), and hemorrhagic shock (severe blood loss).
• Disseminated mucormycosis: fever, malaise (general discomfort), weight loss, anemia (low red blood cell count), leukocytosis (high white blood cell count), organ failure (kidney, liver, heart), septicemia (blood infection), meningitis (brain inflammation), endocarditis (heart valve inflammation), osteomyelitis (bone inflammation), and death.

Pathogenesis in plant hosts: Rots

Rhizopus spp can cause various plant diseases that are characterized by soft rotting of fruits and vegetables. Some of the crops affected by Rhizopus spp include strawberries, tomatoes, sweet potatoes, tobacco, papayas, and stone fruits .

The infection is caused by spores of Rhizopus spp that land on the surface of fruits or vegetables and germinate under favorable conditions of temperature and humidity. The fungi then produce enzymes that degrade the plant cell wall and allow the hyphae to penetrate and colonize the tissues. The fungi also produce toxins that cause tissue necrosis and brown discoloration .

The symptoms of Rhizopus rot vary according to the type of fruit or vegetable infected, but generally include softening, wilting, shriveling, water-soaking, browning, and decay of the affected parts. The fungi also produce black sporangia that are visible on the surface of the rotted tissues .

Rhizopus rot can cause significant losses in post-harvest quality and quantity of fruits and vegetables, especially during storage and transport. The fungi can also spread from infected to healthy fruits or vegetables by contact or by air .

Mucormycosis, also known as zygomycosis, is a rare but serious fungal infection caused by a group of molds called mucormycetes. These molds live throughout the environment and can infect humans and animals through inhalation, ingestion, or direct contact with contaminated surfaces or wounds. Mucormycosis mainly affects people who have weakened immune systems or underlying health conditions, such as diabetes, cancer, organ transplant, burns, or trauma. The infection can affect different parts of the body, depending on the route of exposure and the extent of invasion. The most common forms of mucormycosis are:

• Rhinocerebral mucormycosis: This is the most common form of mucormycosis and involves the infection of the sinuses and the brain. It usually occurs after inhaling fungal spores from the air. The fungus invades the blood vessels and causes tissue necrosis (death) and ischemia (lack of blood flow). The symptoms of rhinocerebral mucormycosis include:

  • One-sided facial swelling
  • Headache
  • Nasal or sinus congestion
  • Black lesions on nasal bridge or upper inside of mouth
  • Fever
  • Vision problems, such as blurred vision, double vision, or eye pain
  • Neurological problems, such as seizures, slurred speech, partial paralysis, or coma

• Pulmonary mucormycosis: This is the second most common form of mucormycosis and involves the infection of the lungs. It usually occurs after inhaling fungal spores from the air or from contaminated medical devices, such as ventilators. The fungus invades the blood vessels and causes tissue necrosis and ischemia. The symptoms of pulmonary mucormycosis include:

  • Fever
  • Cough
  • Chest pain
  • Shortness of breath
  • Hemoptysis (coughing up blood)
  • Respiratory failure

• Cutaneous mucormycosis: This is the third most common form of mucormycosis and involves the infection of the skin and soft tissues. It usually occurs after direct contact with contaminated surfaces or wounds, such as burns, trauma, surgery, or injections. The fungus invades the blood vessels and causes tissue necrosis and ischemia. The symptoms of cutaneous mucormycosis include:

  • Skin redness, warmth, swelling, or pain
  • Blisters or ulcers
  • Black necrotic tissue
  • Skin breakdown or gangrene

• Gastrointestinal mucormycosis: This is a rare form of mucormycosis and involves the infection of the stomach or intestines. It usually occurs after ingesting contaminated food or water. The fungus invades the blood vessels and causes tissue necrosis and ischemia. The symptoms of gastrointestinal mucormycosis include:

  • Abdominal pain
  • Nausea and vomiting
  • Gastrointestinal bleeding
  • Peritonitis (inflammation of the abdominal lining)
  • Bowel infarction (death of bowel tissue)
  • Hemorrhagic shock (severe blood loss)

• Disseminated mucormycosis: This is a rare but life-threatening form of mucormycosis and involves the spread of the infection to multiple organs or systems. It usually occurs in people who have severely compromised immune systems or uncontrolled underlying diseases. The fungus invades the blood vessels and causes tissue necrosis and ischemia in various parts of the body. The symptoms of disseminated mucormycosis depend on which organs are affected, but may include:

  • Fever
  • Malaise (general feeling of illness)
  • Mental status changes or coma
  • Organ failure
  • Death

Mucormycosis is a serious infection that requires prompt diagnosis and treatment. If you have any symptoms that you think are related to mucormycosis, you should contact your healthcare provider immediately.

The definitive diagnosis of mucormycosis requires the laboratory confirmation of the presence and identity of the causative fungi in the clinical specimens. The laboratory methods include direct microscopic examination, histopathological examination, culture, serology, and molecular techniques.

Direct microscopic examination

Direct microscopic examination of the clinical specimens, such as skin scrapings, nasal discharges, bronchoalveolar lavage fluid, or tissue biopsies, can provide a rapid presumptive diagnosis of mucormycosis. The specimens are usually treated with 10-20% potassium hydroxide (KOH) to dissolve the cellular debris and enhance the visibility of the fungal elements. The KOH preparation can be supplemented with fluorescent brighteners such as Blankophor or Calcofluor white, which bind to the fungal cell wall and emit fluorescence under ultraviolet light. However, this method requires a fluorescent microscope.

The characteristic features of mucormycetes that can be observed under the microscope are broad (6-15 µm), ribbon-like, non-septate or sparsely septate hyphae with irregular branching angles, including right-angle (90°) bifurcations. The hyphae may also appear swollen, distorted, or fragmented .

Histopathological examination

Histopathological examination of the tissue sections fixed and stained with hematoxylin and eosin (H&E) or special fungal stains, such as Grocott methenamine-silver (GMS) or periodic acid-Schiff (PAS), can reveal the invasion of blood vessels and tissues by the mucormycetes. The histological features are similar to those seen in direct microscopy, i.e., broad, non-septate or sparsely septate hyphae with right-angle branching .

Histopathology is a very important diagnostic tool since it distinguishes the presence of the fungus as a pathogen in the specimen from a culture contaminant and is indispensable to define whether there is blood vessel invasion.

Culture

Culture of the clinical specimens on appropriate media, such as Sabouraud dextrose agar (SDA) with or without cycloheximide or brain-heart infusion agar (BHI), can isolate and identify the causative species of mucormycetes. The colonies are fast-growing, cottony-fluffy in texture, initially white and then turning grey to yellowish-brown as they mature. The reverse pigmentation is white to pale.

The identification of the species is based on the morphological characteristics of the sporangia, sporangiophores, rhizoids, sporangiospores, and zygospores. Rhizopus spp. produce dark sporangia at the tips of unbranched sporangiophores that arise from root-like rhizoids. The sporangiospores are globose to ovoid, single-celled, hyaline to brown, and striate.

Culture is considered the gold standard for diagnosis of mucormycosis, but it has some limitations. It may take several days to obtain a positive result, it may be negative in cases of prior antifungal therapy or inadequate specimen collection, and it may not differentiate between colonization and infection .

Serology

Serological tests for mucormycosis are based on the detection of antibodies or antigens in the serum or other body fluids of the patients. Various methods have been developed, such as enzyme-linked immunosorbent assay (ELISA), Western blotting, immunohistochemistry, and immunocytofluorimetry. However, these methods are not widely available or standardized and have limited sensitivity and specificity .

Molecular techniques

Molecular techniques for mucormycosis are based on the amplification and sequencing of specific DNA regions of the mucormycetes using polymerase chain reaction (PCR) or other methods. These techniques can provide rapid and accurate identification of the species and subtypes of mucormycetes from clinical specimens or cultures. However, these techniques are also not widely available or standardized and require specialized equipment and expertise .

Automated systems

Automated systems for fungal identification are based on biochemical or proteomic methods that analyze the metabolic or protein profiles of the fungi from cultures. Examples of these systems are VITEK 2, MALDI-TOF MS, and BD Phoenix. These systems can provide rapid and reliable identification of the common species of mucormycetes, such as Rhizopus oryzae, Rhizopus microsporus, and Rhizopus arrhizus. However, they may not be able to identify rare or novel species of mucormycetes and may require further confirmation by molecular techniques .

Mucormycosis is a serious and potentially fatal infection that requires prompt and aggressive treatment. The main components of mucormycosis treatment are:

• Antifungal therapy
• Surgical debridement of infected tissue
• Treatment of underlying conditions and risk factors
• Adjunctive therapy with hyperbaric oxygen (optional)

Antifungal therapy

The first-line antifungal drug for mucormycosis is liposomal amphotericin B , which is given intravenously at a dose of at least 5 mg/kg per day. Liposomal amphotericin B has a better safety profile and higher tissue penetration than conventional amphotericin B. The duration of antifungal therapy depends on the clinical and radiological response, but it usually ranges from several weeks to months.

The second-line antifungal drugs for mucormycosis are isavuconazole and posaconazole , which are triazole agents that have activity against Mucorales. Isavuconazole can be given intravenously or orally, while posaconazole can be given intravenously or as delayed-release tablets. These drugs can be used as salvage therapy for patients who cannot tolerate or fail liposomal amphotericin B, or as maintenance therapy after induction with liposomal amphotericin B . The recommended doses are 372 mg of isavuconazole three times a day on the first day, followed by 372 mg once a day thereafter; or 300 mg of posaconazole twice a day on the first day, followed by 300 mg once a day thereafter.

Other antifungal drugs, such as fluconazole, voriconazole, and echinocandins, are not effective against mucormycosis and should not be used.

Surgical debridement

Antifungal therapy alone is usually not sufficient to control mucormycosis, especially in cases involving the sinuses, the brain, the lungs, or the skin. Therefore, surgical debridement of infected and necrotic tissue is an essential part of mucormycosis treatment . Surgery should be performed as soon as possible after diagnosis and repeated as needed until all infected tissue is removed. Surgery can reduce the fungal burden, improve blood flow, prevent dissemination, and enhance the efficacy of antifungal drugs. However, surgery may also entail significant morbidity and disfigurement, especially in rhinocerebral mucormycosis. Therefore, surgery should be performed by experienced surgeons in consultation with infectious disease specialists and other relevant specialists.

Treatment of underlying conditions and risk factors

Mucormycosis often occurs in patients with underlying conditions or risk factors that impair their immune system or increase their susceptibility to fungal infections. These include diabetes mellitus, ketoacidosis, malignancies, organ transplantation, immunosuppressive therapy, renal failure, cirrhosis, iron overload, and trauma . Therefore, treatment of mucormycosis should also address these underlying conditions and risk factors by optimizing glycemic control, correcting acid-base imbalance, reducing immunosuppression, managing renal dysfunction, chelating excess iron, and treating wounds . These measures can improve the host defense against mucormycosis and reduce the mortality rate.

Adjunctive therapy with hyperbaric oxygen

Hyperbaric oxygen therapy (HBOT) is the administration of oxygen at high pressure to increase the oxygen concentration in the blood and tissues. HBOT has been proposed as an adjunctive therapy for mucormycosis based on its potential benefits such as enhancing the activity of antifungal drugs, inhibiting the growth of Mucorales, improving tissue oxygenation and healing, and modulating the immune response . However, the evidence for HBOT in mucormycosis is limited and conflicting, and there are no randomized controlled trials to support its use . Moreover, HBOT has some drawbacks such as high cost, limited availability, logistical challenges, and possible adverse effects such as barotrauma, oxygen toxicity, and claustrophobia . Therefore, HBOT is not routinely recommended for mucormycosis, but it may be considered as an individualized option for selected patients who have refractory or extensive disease, or who cannot undergo surgery or antifungal therapy . HBOT should be administered in conjunction with standard treatment, not as a substitute .

Rhizopus is a genus of fungi that includes both saprophytic and parasitic species. They can grow on a wide range of organic substrates, such as fruits, vegetables, bread, leather, soil, and animal feces. They have a filamentous, branching, and coenocytic (multinucleated) mycelium that produces three types of hyphae: stolons, rhizoids, and sporangiophores. They reproduce both sexually and asexually by producing spores.

There are about 10 species of Rhizopus, of which three are of industrial and medical importance. They are:

• Rhizopus microsporus var. oligosporus: This species is used to produce fumaric acid, lactic acid, and cortisone. It is also used to make tempeh, a fermented soybean product that is popular in Indonesia and other parts of Southeast Asia .
• Rhizopus oryzae (also known as Rhizopus arrhizus): This species is used in the production of lactic acid and cortisone. It is also used for alcoholic fermentation in some parts of Asia and Africa . It has the ability to biosorb (passively adsorb) heavy metals from contaminated water or soil.
• Rhizopus stolonifer (also known as black bread mold): This species causes fruit rot on strawberry, tomato, sweet potato, and other crops. It is used in the commercial production of fumaric acid, lactic acid, and cortisone . It can also produce biotin, a vitamin that is essential for human health.

These species of Rhizopus have various applications in the food, pharmaceutical, chemical, and environmental industries. They are also important agents of mucormycosis (also known as zygomycosis), a rare but serious fungal infection that affects humans and animals with weakened immune systems .

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