Antibiotic, Probiotic & Lantibiotic

 

Chemotherapeutic agents are chemical substances used for the treatment of infectious diseases or diseases caused by the proliferation of malignant cells. These substances are prepared in the laboratory or obtained from microorganisms and some plants and animals. In general, naturally occurring substances are distinguished from synthetic compounds by the name antibiotics. Some antibiotics are prepared synthetically but most of them are produced commercially by biosynthesis. Microorganisms producing the largest number of useful antibiotics belong to the genera Bacillus, Penicillium, Streptomyces and Cephalosporium in addition to other chemotherapeutic agents like sulfonamides, nitrofurantoin, etc.

Criteria in choosing a useful chemotherapeutic agent:

1.     It must demonstrate selective toxicity for the disease agent.

2.     The host should not become allergic (hypersensitive) to the drug.

3.     The host should not destroy, neutralize or excrete the drug too rapidly.

4.     The organism should not readily become resistant to the drug.

5.     The drug should reach the site of infection.

SPECTRUM OF ACTIVITY
NARROW SPECTRUM	BROAD SPECTRUM
Penicillin	Chlorampenicol
Streptomycin	Chlortetracycline
Erythromycin	Demeclocycline
Lincomycin	Oxytetracycline
Polymyxin B	Tetracycline and derivatives
Colistin	Ampicillin
Vancomycin	Cephalothin
Nystatin	Gentamycin
Spectinomycin	Rifampin
	Tobramycin
	Paromomycin

MODE OF ACTION	ANTIBIOTIC	TARGET
Compete with PABA (para–aminobenzoic acid)	Sulfonamides	Enteric urinary tract infection
	PAS (para–aminosalicylic acid)	Mycobacterium tuberculosis
	Trimethoprim	Broad spectrum activity
	Compete with pyridoxine	Mycobacterium tuberculosis
Disrupt cell membrane	Polymyxins	Gram negative bacteria
	Polyene antibiotics (Nystatin, Amphotericin B)	Fungi
Inhibit cell wall peptidoglycan synthesis	Penicillin	Mostly Gram (+) bacteria
	Cephalosporin
	Bacitracin
	Vancomycin
	Ristocetin
Inhibit RNA synthesis	Rifampin	Gram (+) bacteria
Inhibit DNA synthesis	Mitomycin & Actinomycin
	Nalidixic acid	Gram (–) bacteria of UTI etiology
	Novobiocin	Gram (+) bacteria
	Griseofluvin	Fungi
Inhibit purine synthesis	Trimethoprim	Broad spectrum antibiotic
Inhibit protein synthesis	Chloramphenicol	Broad spectrum antibiotic mostly gram (+) bacteria
	Macrolide antibiotics (Erythromycin, Olendomycin Carbomycin, Spiramycin    Lincomycin, Clindamycin Tetracycline, Streptomycin)

Some organisms develop a tolerance for a new environmental condition and these organisms are referred to as drug fast or drug resistant. Drug resistance may be due to a pre–existing factor in microorganisms or it may be due to some acquired factors.

Emergence of drug resistance can be minimized by

1.  Maintaining sufficiently high levels of the drug in the tissues to inhibit both the original population and first step mutant.

2.  Simultaneously administer two drugs that do not give cross resistance, each of which delays the emergence of mutant resistant to the other drug.

3.  Avoiding exposure to a particularly valuable drug by restricting its use, especially in hospitals.

Some the of Antibiotic Resistant (ABR) Bacteria:

1.     MRSA – Methicillin–Resistant Staphylococcus aureus

2.     VRE – Vancomycin–Resistant Enterococcus

3.     CRE – Carbapenem–Resistant Enterobacteriaceae (CRE)

4.     CRAB – Carbapenem resistant Acinetobacter baumannii

5.     MDR–TB – Multi–Drug–Resistant Mycobacterium tuberculosis

Except for Mycobacterium, most of the bacteria enumerated belongs to ESKAPE bacteria group which are a group of opportunistic pathogens consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species characterized by increased levels of resistance towards multiple classes of first line and last–resort antibiotic.

ESKAPE pathogens are mainly responsible for nosocomial infections and these infections are defined as hospital–acquired infections (HAIs) that affect patients within 48 hours of admission or 3 days of discharge or 30 days of an operation. No literature is available regarding the exact characteristics that classify bacteria under ESKAPE.

ESKAPE pathogens are resistant to oxazolidinones, lipopeptides, macrolides, fluoroquinolones, tetracyclines, β-lactams, β-lactam–β-lactamase inhibitor combinations, and last-line antibiotics including carbapenems, glycopeptides, and polymyxins.

The mechanisms of multidrug resistance exhibited by ESKAPE are broadly grouped into three categories namely:

1.  Drug inactivation is commonly caused by an irreversible cleavage catalyzed by an enzyme.

2.  Modification of the target site where the antibiotic may bind.

3.  Reduced accumulation of drug either due to reduced permeability or by increased efflux of the drug.

They are also able to form biofilms that physically prevent the immune response cells of host as well as antibiotics to inhibit the pathogen. Moreover, biofilms protect specialized dormant cells called persister cells that are tolerant to antibiotics which cause difficult-to-treat recalcitrant infections.

 

THE LANTIBIOTICS

Bacteriocins are antimicrobial substances produced by lactic acid bacteria (LAB), including organic acids, hydrogen peroxide, diacetyl, and inhibitory enzymes. The bacteriocin producer expresses immunity proteins to protect themselves from the bacteriocin they produce, and little was known about these proteins.

Lantibiotics are peptide-derived antimicrobial agents that are produced by the ribosomes and post–translationally modified to their biologically active forms.

Classification of Bacteriocins:

1.  Class I bacteriocins (lantibiotics) are small (<5 kDa) peptides containing the unusual amino acids lanthionine (Lan), β-methyllanthionine (MeLan) and several dehydrated amino acids. They are heat–stable and mainly induce destabilization and permeabilization of the bacterial membranes or cause pore formation into the membrane.

Examples include Nisin, Lacticin 481, and the two-component lantibiotics such as cytolysin produced by Enterococcus faecalis, lacticin 3147 produced by L. lactis, and staphylococcin C55 produced by Staphylococcus aureus.

2.  Class II bacteriocins are small thermostable peptides with an amphiphilic helical structure that allows for their insertion in the cytoplasmic membrane of the target cell, thereby promoting membrane depolarization and cell death.

a.  Subclass IIa – often designated as pediocin–like bacteriocins, constitutes the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization (e.g., Pediocin PA-1, Sakacin A, and Enterocin A).

b.  Subclass IIb – the activity of these bacteriocins depends on the complementary activity of two peptides. The combined effect of the two peptides of these bacteriocins is much greater than the total activity calculated from the individual effect of these peptides (e.g., Lactococcin G, Lacticin F).

c.   Subclass IIc – also known as “Circular Bacteriocins.” They consist of the cyclic bacteriocins whose N– and C–termini are covalently linked, and the circular molecule is resistant to several proteases and peptidases (e.g., Acidocin B).

d.  Subclass IId – contains the one-peptide non-cyclic bacteriocins that show no sequence similarity to the pediocin-like bacteriocins.

3.  Class III are large (>30 kDa) heat-labile proteins that are capable of degrading cell wall murein. This group includes some colicins, zoocins, megacins (Bacillus megaterium), klebicin (Klebsiella pneumonia), helveticins I and J (Lactobacillus helveticus), and enterolysin A (Enterococcus faecalis). The new class III bacteriocin BLF3872 from LF3872 has not yet been studied.

4.  Class IV, the “complex bacteriocins” has also been suggested, which require non-proteinaceous moieties for activity. This class, however, has not been sufficiently studied at the biochemical level.

Studies on the genetics and biochemistry of bacteriocins have principally focused on members of Class I and II, due to the abundance of these peptides and their potential commercial applications.

THE PROBIOTICS

Probiotics are live microorganisms that are intended to have health benefits when consumed or applied to the body. They can be found in yogurt and other fermented foods, dietary supplements, and beauty products. The most common are bacteria that belong to groups called Lactobacillus and Bifidobacterium. Other bacteria may also be used as probiotics, and so may yeasts such as Saccharomyces boulardii.

Prebiotics are food supplements that are nondigestible by the host but can exert beneficial effects by selective stimulation of growth or activity of microorganisms that are present in the intestine.

For a potential probiotic strain to exert its beneficial effects, it is expected to exhibit certain desirable properties. The ones currently determined by in vitro tests are:

1.  Acid and bile tolerance which seems to be crucial for oral administration,

2.  Adhesion to mucosal and epithelial surfaces, an important property for successful immune modulation, competitive exclusion of pathogens, as well as prevention of pathogen adhesion and colonisation,

3.  Antimicrobial activity against pathogenic bacteria,

4.  Bile salt hydrolase activity.

There is increasing evidence in favor of the claims of beneficial effects attributed to probiotics, including improvement of intestinal health, enhancement of the immune response, reduction of serum cholesterol, and cancer prevention. These health properties are strain specific and are impacted by the various mechanisms mentioned above. While some of the health benefits are well documented others require additional studies to be established. In fact, there is substantial evidence to support probiotic use in the treatment of acute diarrheal diseases, prevention of antibiotic-associated diarrhea, and improvement of lactose metabolism, but there is insufficient evidence to recommend them for use in other clinical conditions.

Recent evidence suggests that exposure to bacteria in early life may exhibit a protective role against allergy and in this context, probiotics may provide safe alternative microbial stimulation needed for the developing immune system in infants.

Evidence also suggests that food products containing probiotic bacteria could possibly contribute to coronary heart disease prevention by reducing serum cholesterol levels as well as to blood pressure control.

The Free-Living Amoeba

 

The classical taxonomic classification divided the Protozoa into four groups:

a.     Sarcodina (amoebae)

b.     Mastigophora (flagellates)

c.      Sporozoa (most parasitic protozoa)

d.     Infusoria (ciliates)

This taxonomy has been totally abandoned by the International Society of Protozoologists based on modern morphological approaches such as biochemical pathways and molecular phylogenetics (e.g., 18S rRNA sequences). The older hierarchical systems consisting of the traditional “kingdom,” “phylum,” “class,” “subclass,” “super-order,” “order,” has been replaced by a new vocabulary.

According to this new schema, the Eukaryotes have been classified into six clusters or “Super Groups,” namely:

a.     Amoebozoa

b.     Opisthokonta

c.      Rhizaria

d.     Archaeplastida

e.     Chromalveolata

f.      Excavata

The three amoebae that are dealt within this article have been classified under two Super Groups, Amoebozoa and Excavata, as follows:

a. Acanthamoeba and Balamuthia are classified under Super Group Amoebozoa: Acanthamoebidae

b. Naegleria fowleri under Super Group Excavata: Heterolobosia: Vahlkampfiidae

This schema has been proposed as the basis for future revisions.

For educational purposes, the classic phylogeny of the parasite was described here. And unlike amoeba belonging to Phylum Sarcodina that requires host to survive, parasites belonging in this group are free living (i.e., doesn’t require host to survive).

NAEGLERIA FOWLERI

Phylum Percolozoa

Subphylum Tetramitia

Order Schizopyrenida

Family Vahlkampfiidae

Genus Naegleria

Naegleria fowleri is an amphizoic amoeba, as it can survive in a free-living state in water, soil, or in the host, which can be the human central nervous system (CNS) and causes a disease known as Primary Amebic Meningoencephalitis (PAM) thus its reputation as "brain-eating amoeba."

The initial symptoms of PAM are indistinguishable from bacterial meningitis, while the symptoms of GAE can mimic a brain abscess, encephalitis, or meningitis.

The amoeboid stage is roughly cylindrical, typically around 20–40 μm in length. They are traditionally considered lobose amoebae, but are not related to the others, and unlike them, do not form true lobose pseudopods. Instead, they advance by eruptive waves, where hemispherical bulges appear from the front margin of the cell, which is clear. The flagellate stage is slightly smaller, with two or four anterior flagella anterior to the feeding groove.

Naegleria fowleri has been thought to infect the human body by entering the host through the nose when water is splashed or forced into the nasal cavity. Infectivity occurs first through attachment to the nasal mucosa, followed by locomotion along the olfactory nerve and through the cribriform plate (which is more porous in children and young adults) to reach the olfactory bulbs within the CNS. Once Naegleria fowleri reaches the olfactory bulbs, it elicits a significant immune response through activation of the innate immune system, including macrophages and neutrophils. Naegleria fowleri enters the human body in the trophozoite form. Structures on the surface of trophozoites known as food cups enable the organism to ingest bacteria, fungi, and human tissue. In addition to tissue destruction by the food cup, the pathogenicity of Naegleria fowleri is dependent upon the release of cytolytic molecules, including acid hydrolases, phospholipases, neuraminidases, and phospholipolytic enzymes that play a role in host cell and nerve destruction. The combination of the pathogenicity of Naegleria fowleri and the intense immune response resulting from its presence results in significant nerve damage and subsequent CNS tissue damage, which often result in death.

The quickest way to diagnose Naegleria fowleri infection is by microscopic examination of fresh, unfrozen, unrefrigerated cerebrospinal fluid (CSF).

Both chlorinated and salt water significantly decrease the risk of Naegleria fowleri infection due to its inability to survive in such environments. Thus, avoidance of exposure to freshwater bodies such as lakes, rivers, and ponds, especially during the summer months when the water temperature is higher.

ACANTHAMOEBA SPP.

Phylum Amoebozoa

Class Conosea

Order Centramoeba

Family Acanthamoeba

Some of the pathogenic species:

a.     Acanthamoeba castellanii

b.     Acanthamoeba culbertsoni

c.      Acanthamoeba polyphaga

d.     Acanthamoeba healyi

e.     Acanthamoeba divionensis

Diseases caused by Acanthamoeba include keratitis and granulomatous amoebic encephalitis (GAE).

Acanthamoeba keratitis (AK) is associated with trauma to the cornea or contact-lens wear and the use of amoeba–contaminated saline. Minor erosion of the corneal epithelium may occur while wearing hard or soft contact lenses, and the subsequent use of contaminated saline solution is the major risk factor for Acanthamoeba keratitis. AK is characterized by inflammation of the cornea, severe ocular pain, and photophobia, a characteristic 360^o or paracentral stromal ring infiltrate, recurrent breakdown of corneal epithelium, and a corneal lesion refractory to the commonly used antibiotics. Typically, only one eye is involved; however, bilateral keratitis has also been reported. It is the MBP (mannose binding protein) that mediates the adhesion of the amoeba to corneal epithelial cells and is central to the pathogenic potential of Acanthamoeba.

A unique and characteristic feature of Acanthamoeba spp. is the presence of fine, tapering, thorn-like acanthopodia that arise from the surface of the body.

a. The trophozoites range in size from 15 to 50mm depending upon the species. They are uninucleate, and the nucleus has a centrally placed, large, densely staining nucleolus. The cytoplasm is finely granular and contains numerous mitochondria, ribosomes, food vacuoles, and a contractile vacuole. When food becomes scarce, or when it is facing desiccation or other environmental stresses, the amoebae round up and encyst.

b. Cysts are double-walled and range in size from 10 to 25mm. Cysts are uninucleate and possess a centrally placed dense nucleolus. Upon return to favourable growth conditions, the dormant amoeba is activated to leave the cyst by dislodging the operculum and reverting to a trophic form

(1)   The outer cyst wall, the ectocyst, is wrinkled with folds and ripples and contains protein and lipid.

(2)   The inner cyst wall, the endocyst, contains cellulose and hence is Periodic Acid Schiff (PAS) positive. The endocyst varies in shape: it may best ellate, polygonal, oval, or spherical.

(3)   Pores or ostioles that are covered by convex–concave plugs or opercula are present at the junction of the ectocyst and the endocyst.

In either the trophic or the cyst stage these organisms have a wide distribution in nature, and it is virtually impossible not to isolate members of this genus from soil, water, and other samples.

Acanthamoeba spp. Are ubiquitous and occur worldwide. They have been isolated from soil, fresh and brackish waters, bottled mineral water, cooling towers of electric and nuclear power plants, heating, ventilating and air conditioning units, humidifiers, Jacuzzi tubs, hydrotherapy pools in hospitals, dental irrigation units, dialysis machines, dust in the air, bacterial, fungal and mammalian cell cultures, contact-lens paraphernalia, ear discharge, pulmonary secretions, swabs obtained from nasopharyngeal mucosa of patients with respiratory complaints as well as of healthy individuals, maxillary sinus, mandibular autografts, and stool samples. In addition, several Acanthamoeba species have been isolated from the brain, lungs, skin, and cornea of infected individuals.

BALAMUTHIA MANDRILLARIS

Balamuthia mandrillaris is a free-living amoeba that is found in the soil and fresh water and is associated with Granulomatous Amoebic Encephalitis (GAE), a “brain-eating” disease both in humans and animals. Symptoms of granulomatous amebic encephalitis begin gradually. Confusion, headache, and seizures are common. People may have a low-grade fever, blurred vision, changes in personality, and problems with speaking, coordination, or vision. One side of the body or face may become paralyzed.

Balamuthia mandrillaris may cause skin sores in addition to the symptoms above. Most infected people die, usually 7 to 120 days after symptoms begin.

Possible modes of transmission of Balamuthia include inhalation and inoculation through broken skin.

Balamuthia mandrillaris, like Acanthamoeba, has only two life-cycle stages, namely the vegetative trophozoite and the dormant cyst.

a. The trophozoite is pleomorphic and measures from 12 to 60 µm (mean of 30 µm). The trophic amoebae are usually uninucleate, although binucleate forms are occasionally seen. The nucleus contains a large, centrally placed, dense nucleolus; occasionally, however, amoebae with two or three nucleolar bodies have been seen, especially in infected tissues.

b. Cysts are also uninucleate, are spherical, and range in size from 12 to 30 µm (mean of 15 µm). Cysts, when examined with a light microscope, appear to be double walled, the outer wall being wavy and the inner wall round, and pores are not seen in the wall. Ultrastructurally, however, the cyst wall has three layers:

(1)   an outer thin and irregular ectocyst,

(2)   an inner thick endocyst, and

(3)   a middle amorphous fibrillar mesocyst.

In general, Acanthamoeba spp. and Balamuthia are difficult to differentiate in tissue sections by light microscopy because of their similar morphology. However, they can be differentiated by immunofluorescence analysis of the tissue sections using rabbit anti–Acanthamoeba or anti–B–mandrillaris sera.

While Balamuthia and Naegleria share some similarities, Balamuthia is more difficult to detect. This is due to its resemblance to histiocytes under the microscope and unique culture requirements. Unlike Naegleria, Balamuthia cannot be grown on agar because it only feeds on mammalian cells and other amoebas. Furthermore, healthy individuals can be seropositive for Balamuthia antibodies due to the amoeba’s pervasive presence in the environment, while those with GAE show low titers. Additionally, cerebrospinal fluid analysis rarely demonstrates the organism, and the time course for the appearance of lesions and the onset of GAE is inconsistent. Balamuthia, unlike most of other free-living amoebae, does not feed on Gram-negative bacteria and therefore the use of non-nutrient agar coated with bacterial cultures has resulted to be ineffective for its growth.

These amoebae were normally cultured on monolayers of African green monkey kidney cells. Upon axenic cultivation, amoebae grew at various temperatures ranging from 25°C to 37°C (optimal growth at 37°C) and remained viable for up to several months, but they became smaller over time. In contrast, mammalian cultures can be used persistently as feeder cells to culture Balamuthia amoebae over longer periods, without any modifications in their general appearance. All tested cell cultures, including human brain microvascular endothelial cells (HBMEC), human lung fibroblasts, monkey kidney (E6) cells, and African green monkey fibroblast-like kidney (Cos-7) cells, supported the growth of B. mandrillaris.