PHAR2921 Weeks 1-4 PDF

PHAR2812 – Infectious disease Bacteroidetes How are Bacteria Classified? They are highly abundant in intestines and are Phenetic (Phenotype) responsible for polysaccharide degradation of - Gram reaction and morph...

PHAR2812 – Infectious disease Bacteroidetes How are Bacteria Classified? They are highly abundant in intestines and are Phenetic (Phenotype) responsible for polysaccharide degradation of - Gram reaction and morphology non – digestible carbohydrates. E.g., - Carbon sources and energy sources Bacteroides.X - Electron acceptors (e.g., - Obligate anaerobe, heterotrophic, gram- aerobic/anaerobic) negative rods ↳ cannot produce own food - Normal flora can be opportunistic Phylogenetic (genetic) pathogens The study of evolutionary relationships among - The most abundant cell in the human biological entities – often species, individuals body or genes. It is a systematic approach. - Several beneficial species; digestion of - Ribosomal RNA sequence carbohydrates, exclude pathogens by - Other DNA, RNA, and protein sequence competition (salmonella) Phenotypes are important for identification or D B. Fragilis pathogenicity. The opportunistic pathogen causes infection if - In some cases, systemics agree with it escapes the gut e.g., abscesses, phenotype e.g., all endospore formers septicaemia, appendicitis. are Gram-positive. - Virulence factors - In other cases, the classifications bloodpoisoning o Capsule, antibiotic resistance disagree e.g., anaerobic growth occurs in diverse bacteria. Phylogenetic tree of Bacteria Spirochaetes Spirochaetes, a phylum that includes medically important pathogens such as the causative agents of Lyme disease, syphilis, and leptospirosis, are in many ways highly unique bacteria. E.g., Treponema Q Treponema Diverse Gram – Negative bacteria can cause - Anerobic, heterotrophic, gram-negative, disease spirochaetes. Proteobacteria - Obligate parasites require animal cells It is an important phylum of gram-negative for growth bacteria. It includes a wide variety of pathogenic genera which are also responsible for nitrogen fixing. E.g., - Escherichia -Ncycle - Salmonella - Vibrio Use axial filament structure and corkscrew - Pseudomonas motility which is great for movement through - Neisseria viscous fluid. - Rickettsia  Treponema – T. Palladium Symptoms This causes syphilis – which is a sexually - Diarrhea transmitted disease (STD) - Fever - Effects 10 chancres, secondary rash, released by bacteria tertiary → nervous system damage. Virulence Factors toxin - Can't be grown in standard media - Endotoxin (all G-) can induce - Degenerate small genome (1 Mb) inflammation and fever as an immune response in higher organisms. Reaction to endotoxins can lead to anaphylactic B Escherichia E. coli is a type of bacteria that normally lives shock and death of patients. inside our intestines, where it helps the body - Enterotoxin (some) is a substance that break down and digest food. is harmful to your digestive system. Some types (or strains) of E. coli, though, are infectious (causing infections that can spread ⑤ Salmonella Facilitative anaerobic, heterotrophic, gram- to others). E.g., Salmonella, Klebsiella, negative rods. Yersinia. - Normal flora in animal gut, pathogenic to humans - Motile by peritrichous flgae - S. enterica; foodborne infection, self– limiting diarrhoea ↓ - S. typhi; water-borne, typhoid fever, potentially fatal Virulence factors. - Enterotoxin - Cytotoxin - kill cells Identifying Enterobacteriaceae -G-ve Selective + Differential agars for identifying Enterobacteriaceae ⑨E. coli - Most Strains are normal flora, and beneficial e.g., E.coli biosynthesizes - vitamin K Some strains are pathogenic; these are & Vibrio Facultative anaerobic, heterotrophic, gram- usually food or water-borne negative curved rods. - O157 – infection by animal food, O111 - Motile, various flagella arrangements - Habitat is primarily marine, but can cause gut infection.  V. Cholerae The most pathogenic Vibrio species - Causes cholera; severe diarrhoea → transmission (usually by faecal contamination of water) Virulence Factor; - Cholera toxin (an exotoxin) - Common in sewage contamination - Rarely a food – borne disease (may come from seafood) Virulence factors - Capsule; evasion of immune response ⑦ Pseudomonas Aerobic, heterotrophic, gram-negative rods, - Fimbriae; adhesion to tissues motile (polar flag). Neisseria – N. meningitidis - Large genome (6Mb) → Metabolically Meningitis: inflammation of the meninges versatile (membrane around the brain) it causes fever, headache, confusion and death. P. aeruginosa from hospital - Serious, rapidly progressing disease – ~ Nosocomial infection (hospital-acquired) needs rapid diagnosis and treatments especially burns. (antibiotics) - Analysis; microscopic examination of Virulence factors CSF - Innate antibiotic resistance (low membrane permeability), haemolysin, proteases. Lipids proteins that lys is , RBC - These infections are common, however, person–person transmission is rare… E.g., Nosocomial infection – P. aeruginosa is transmitted from nurse fingernails to many heart surgery patients. - Metabolic versatility helps P.aerug to Neisseria - N. gonorrhoea colonise diverse niches (e.g., - Gonorrhoea; a sexually transmitted fingernails, heart) disease - Diagnose by microscopic examination - Safranin: epithelial cells and Neisseria ⑨Neisseria appear pink. - Note adherence of Neisseria cells to the Aerobic, heterotrophic, gram-negative diplococci. epithelial - Habitat; mammalian mucous membranes - Carrier; back of throat, nose Pathogenic Species - N. gonorrhoea – causes gonorrhoea - N. meningitides – causes meningitis; these cells adhere to cilia in the respiratory tract  Human body louse Pediculus humanus is a vector for spreading R. prowazekii Diversity of bacteria – Gram positives Phylogenetic tree of the bacteria ⑨ Rickettsia Aerobic, heterotrophic, gram-negative, coccobacilli. - Cannot be grown in vitro – only in tissue culture - Bacteria are very dependent on host metabolism - Intracellular parasites of arthropods – e.g., fleas, lice, ticks - Small degenerate genome (1 Mb) → Both gram-positive and negative bacteria are specialised lifestyle medically important - Related to mitochondrion Rickettsia is inside a eukaryotic cell (from tick). Light micrograph, with carbol duschon stain. ⑩ Chlamydia - Aerobic, heterotrophic, gram-positive Rickettsia is transmitted via bites or faeces of cocci. arthropods → various fever diseases. - Obligate intracellular parasites of - R. prowazekii → Epidemic typhus humans and animals o Headache, fever, rash and up to - Causes sexually transmitted disease 50% mortality and infection o Overcrowded conditions; - Cannot be grown on agar, small genome transmitted by body louse (1 Mb) consistent with host dependence - Virulence factors - An energy parasite – dependent on host o Adhesin cells for ATP and other metabolites o Phospholipase Chlamydia in eukaryotic cells x7000 o Lactobacillus; normal flora o Bacillus and clostridium; are primarily soil organisms – some are pathogens. Actinobacteria; aerobic, gram-positive rods or filaments; some make exospores (e.g. streptomyces) - Streptomyces – makes antibiotics - Mycobacterium – causes tuberculosis C.trachomatis – Chlamydia Causes urethritis (STD) and trachoma (eye Streptomyces colonies have; infection). - Powdery surface - Diffusible pigments Bacillus - Facultative anaerobic, heterotrophic, gram-positive rods - Ubiquitous in environment especially in soil (Ubiquitous means found everywhere) - Transient flora on the skin - Older / starving / stressed cells make endospores. Virulence factors - An unusual cell wall allows growth B. anthracis inside phagocytes - Has no peptidoglycan → Intrinsic - Causes Anthrax, highly infectious and resistance to all antibiotics targeting PG. deadly disease - Usually zoonotic; transmitted from Peptidoglycan: provides rigidity to the cell animals (cattle and sheep…) wall; the cell walls of Gram-positive bacteria - Possible bioterrorism agent due to may contain up to 40 layers of peptidoglycan, stable endospores conferring significant mechanical strength. - Plasmid–encoded virulence factor; exotoxin Gram-positive bacteria Phylum firmicutes, phylum actinobacteria - Distinguished from gram negatives by thicker peptidoglycan and lack of outer membrane - Divided into firmicutes (Low GC) and actinobacteria (high GC) groups - GC means the percentage of GC content in DNA “Anthrax” can be present in three different ways Firmicutes; facultative anaerobic rods or - Cutaneous; more common causing 20% cocci; some make endospores. mortality - Gram positive - Gastrointestinal; rare causes 50% - May be facultatively anaerobic or strictly mortality anaerobic (can live without O2). - Pulmonary; rare causing 80% mortality - Contain both normal flora and pathogenic species All are potentially treatable with antibiotics Lactobacillus e.g., ciprofloxacin. L. Acidophilus - Facultative anaerobic, heterotroph, gram-positive non – sporing rods - Normal flora of the mouth, gut, and vagina i.e., non – pathogenic - Probiotic effect? Competition with pathogens in gut - Cheese + Yoghurt production; lactic acid fermentation Clostridium - Anaerobic, heterotrophic, gram positive Gram – positive cocci rods with endospores - All are non – motile and either aerobic or - Habitat; soil, human and animal gut facultative - Some species are normal flora, some - Contains both normal flora and are pathogens pathogenic species Pathogenic clostridia’s make potent exotoxins - Medicaly important genera; - C. Tetani → tetanus - C. botulinum → Botulism C. Tetani - Tetanus; muscle rigidity, death due to respiratory failure - Virulence factor: an exotoxin – tetanospasmin (a neurotoxin that blocks the transmission of relaxation signals to muscles causing body rigidity → death Staphylococcus from respiratory paralysis) - Facultative anaerobic, heterotrophic, - Requires deep wounds to multiply gram-positive clumps - Common source: soil/manure - Habitat; human-associated either as inoculated into puncture wound – not a normal flora (skin, nose) or pathogens contagious disease (wound infection) - Tough cell wall makes staph resistant to C. Botulinum physical stress (desiccation) → long - Causes botulism; a dangerous type of survival in environment food poisoning (common–source - Resistant to salt – trait needed to survive disease) on skin and nose - Lives in the soil; spores enter food chain, persist through processing and germinating in food (anaerobic, neutral pH) - Virulence factor; botulinum toxin (BOTOX); the deadliest poison known, lethal dose (1 mcg) S. Aureus “Golden Staph” - Aerobic, heterotrophic, gram-positive - Opportunistic pathogen rods - Infections; wound infections, boils, - Mycolic acids In cell wall → creates impetigo, toxic shock waxy layer → “Acid Fast” stain - Nosocomial spread – hospital strains - Wax protects against stresses including antibiotic-resistant the immune system - Morphology arrangement – staph vs - Pathogenic species; tuberculosis (TB) strep and M. leprae (leprosy) - Virulence factors; coagulase, exotoxins - Coagulase as a diagnostic test M. Tuberculosis - The obligate pathogen of humans – no environmental reservoir - One of the “Big 3” killer diseases of humanity - Chronic infection of the lungs; fever, coughing and weight loss - Can be grown in vitro – but slow growing and fastidious Streptococcus - Virulence factors; waxy cell wall gives - Facultative anaerobic, heterotrophic, resistance to many stresses e.g., gram-positive cocci in chains antibiotics and macrophages. - Habitat; mouth and gut of animals and (Obligate pathogen means requires a host to humans survive) Beneficial streptococci Putting it altogether - S. thermophilus; yoghurt + cheese production – normal flora Harmful streptococci - S.pyogenes; scarlet fever – contagious and mostly affects young children - S. pneumoniae; pneumonia S. pneumoniae - Can cause pneumonia and meningitis - Characteristics; alpha – haemolysis reaction on blood agar - Virulence factors; capsule, haemolysin; Microbiology and Infection Medically encapsulated strains are 100,000 times important viruses more virulent. Viruses are a major cause of acute and chronic infections – which can lead to asymptomatic, mild and severe disease. Risk factor for other diseases such as dementia, and cancer. Multiple sclerosis, cirrhosis, autoimmunity and diabetes. Mycobacterium Transmission American biologist David Baltimore further - Direct contact categorised viruses based on; - Sexual 1) The from of nucleic acid in the virion - Vertical (maternal) and; - Insect borne 2) The relationship of the viral genome to - Faecal production of mRNA (and therefore - Drug use (needle sharing) proteins) o dsDNA What are viruses? o ssDNA They are small infectious agents that cause o dsRNA disease in humans – it can infect organisms in o +ssRNA all kingdoms of life and require a host to o -ssRNA replicate. o ssRNA-RT - Lack machinery for making proteins and o dsDNA-RT cannot generate energy - Composed of lipids, proteins and Viral enzymes such as reverse transcriptase, carbohydrates RNA polymerase, proteases are prominent - Possess a nucleic acid genome (DNA or targets for anti-viral drugs as these enzymes RNA); utilise a genetic code. share little, if any homology to cellular proteins. When viruses replicate all the components Influenza virus types used to assemble progeny are freshly The types of influenza cause upper respiratory synthesised. This is referred to as de novo tract infections in humans with fever of varying replication. severity and gastrointestinal tract infections in some animals. Components of a virion. - Symptoms usually last three weeks All viruses - Seasonal outbreaks occur during winter - Genome (DNA or RNA) when the humidity and ambietnt - Nucleocapsid temperature are optimal for the viability - Capsid of virus particles. - Surface proteins Influenza A (IAV) Some classes of viruses - Endemic to many species (humans, - Protein enzymes necessary for birds, pigs, horses and bats) replication - Named after neuraminidase and - Tegument/ matrix protein - Haemagglutinin subtypes - Envelope Influenza B Baltimore Classification - Endemic to humans and responsible for some mortalities but usually less severe than type A Influenza C - Endemic to humans – cause of only mild illness or asymptomatic Influenza D - Not known to infect humans  Mutations in NA can lead to resistance to NA inhibitors. From one season to the next, resistance can be seen to increase dramatically (80%). Seasonal epidemics Seasonal flu - Caused by human types A, B and C which infext 5 – 20% of the population each yeae of which 0.1% are Entry of influenza virus hospitalised. Entry is mediated by trimers of HA of the virus - There is a 1 in 10,000 mortality rate; surface binding sialic acid on the surface cells. mostly in the elderly and the young. - Sialic acid is found ubiquitously on surface glycoproteins throughout the The best protection is via an inactivated human body vaccine administered by an intramuscular - Its entry is restricted to the upper injection. The 2021 quadrivalent vaccine respiratory tract by the availability of consists of; proteases that are required to process HA into an active form. Anti – viral against influenza virus Influenza and SARS-Cov-2 Genome replication and gene expression NSW Immunisation Schedule includes immunisation against the following; - Hepatitis B - Polio - Rotavirus - Measles - Mumps - Rubella - Varicella - HPV Control of influenza by antivirals; targeting - Influenza NA Neuraminidase inhibitors are antiviral drugs Seasonal epidemics and pandemic flu that are effective against influenza virus. They Pandemic flu include oseltamivir (Tamiflu) and Zanamivir - Caused by zoonotic spillover which an (Relenza) and are effective against Influenza A animal IAV (often avian) is introduced and B. These drugs are structural mimics of into the human population sialic acids. - It adapts quickly to efficiently transmit between human hosts Pandemics may introduce novel or What are Protists? substantially divergent HA and NA stereotypes They are Eukaryal microbes that are not fungi. to humans for which there is little to no pre- Algae = Photosynthetic protists existing immunity. Protozoa = chemoorganotrophic protists - Amoebae The emergence of pandemics is unpredictable, - Flagellates as is the virulence of pandemic strains. The - Parasitic protists 1918 Spanish flu was one of the most devastating infectious disease outbreaks of all time with a case fatality rate of approximately 3% - Causes 50 – 100 million deaths - Many from bacterial co-infections (no antibiotics) Antiviral such as zanamivir have been stockpiled by several countries for the event of future pandemics. Emergence of pandemic influenza; antigenic shift Reassortment of segmented influenza genome to generate new HA and NA stereotypes so no herd immunity. - Occurs when cells become infected with two or more different influenza strains and a mixing of genomic segments occurs. - New flu variants do not transmit readily, a pandemic requires further adaptation for efficient human to human transmission. Medically Important Fungi and Protists Importance of fungi to animal health - Food spoilage fungi and mycotoxins - Allergic fungi and hypersensitivity - Commensal and pathogenic fungi Microbes: Phylogeny and medically important Trypanosomes groups - Sleeping sickness (Africa) The prokaryote ‘microbes’ – Bacteria and - Chagas Disease (America) Archaea. The Eukaryal ‘microbes’ – algae, - Leishmaniasis (World Wide) protozoa and fungi. Complex life cycles with arthropod vectors. Antigenic variation – the glycoprotein composition is switched cyclically meaning the cell has 1 of 1000 options at any one time – vaccines are unrealistic proposition. Few Drugs o Filamentous and Mutualistic (e.g. Available drugs target aspects of metabolism Mycorrhizae) that are of greater importance to parasite cells than host cells. Fungal Nutrition Heterotrophic = energy from organic carbon Apicomplexans sources. All are either inter or intra-cellular parasites of animals. They include: Absorptive (saprophytic) nutrition = soluble - Plasmodium (malaria) nutrients. - Toxoplasma; cryptosporidium - Insoluble nutrient sources. Such as - Eimeria dead plant matter, degraded outside the fungal cell by secreted enzymes Apicomplexans share a unique collection of structures called the apical complex (designed Host associations (Symbiosis Spectrum) to penetrate host cells) and.. - Plant mutualisms common – 70% plants - In addition, many have a unique plastid need mycorrhizae called the apicoplast (thought to be a - Plant parasitism common (fungal plant relict chloroplast). pathogens) - Apicomplexans typically have complex - Animal commensals (rumen fungi, skin life cycles that can involve multiple commensals) hosts. - Animal pathogens. Fungal Morphologies Fungi microbes that are typically multicellular and can grow at a macroscopic scale. What are fungi? - Eukaryotic - Saprophytic heterotrophs Hyphae can produce spores at their tips which allow the fungus to disperse and reproduce. o No chlorophyll - Typically aerobic Fungi can have a large “foraging range” o Some are anaerobic (e.g., saccharomyces and rumen Long Distance nutrient foraging requires bug fungi) size and inter – cellular transport. - Long hyphal filaments require rigid cell - Cell walls common – no peptidoglycan o Chitin, beta – glucans and walls. Eukaryal phylogeny glycoprotein - Reproduce by both asexual and sexual - This is where fungi fit with other methods. organisms. - Fungi are closer to animals than to - Single or multi-celled microbes o Single celled (yeast morphology) plants and most protists o This explains why fungal o Filamentous and Independent (E.g., typical mushrooms) diseases are so difficult to treat. Concept Check Acute toxic phenomena associated with 1) Can Fungi perform photosynthesis? mycotoxins. They cannot perform photosynthesis. They lack chlorophyll which is a necessary pigment for photosynthesis. Fungi obtain their nutrients by breaking down organic matter, making them heterotrophs. 2) What’s the main difference between fungal and bacterial cell walls? Fungal: cell wall made of chitin (a tough, flexible polysaccharide) Bacterial cell: made of peptidoglycan (mesh- like polymer of sugars and amino acids). Mycotoxicosis 3) Which organism’s cell biology Acute processes are closest to humans? - Moderate – high levels toxin Fungi have cell biology closest to humans. They - Specific, observable disease are both eukaryotic cells that have similar cellular processes such as DNA replication, Chronic protein synthesis and metabolic pathways - Low – moderate levels of toxins making them the closest to human cells. - Weight loss, infertility and failure to thrive Importance of Fungi to animal health 1. Allergenic fungi and hypersensitivity Mycotoxin–associated diseases 2. Food spoilage fungi and mycotoxins - Low – very low levels of toxin 3. Commensal and pathogenic fungi - General immunosuppression, increased susceptibility of infectious disease Allergenic Fungi and Hypersensitivity Main genera causing problems: Commensal and pathogenic fungi – the - Aspergillus discipline of medical mycology - Penicillium Invasive mycoses – The big three - Alternaria - Candidiasis - Cladosporium - Aspergillosis - Cryptococcosis These grow under damp conditions and make allergenic spores and volatiles. Superficial, cutaneous and subcutaneous mycoses Food spoilage fungi and mycotoxins - Infect skin and underlying tissue. Fungi can grow and damage all types of foods. This is dangerous as they may make Concept Check mycotoxins which is not safe for eating. 1) What is the difference between mycosis and mycotoxicosis? Mycosis – infection caused by fungi – affects various parts of the body (skin or internal organs) E.g.. athletes’ food ring worm and aspergillosis… The main cause is the direct invasion of fungal organisms into tissues. Mycotoxicosis – poisoning caused by toxins - Economic impact and long fungistatic produced by fungi (mycotoxins) – usually treatment regime absorbed inhaled or ingested. E.g., aflatoxicosis caused by Aspergillus species. Superficial infections The cause is exposure to toxic compounds Disease names produced by fungi rather than an infection. - Seborrheic dermatitis (severe dandruff) o Sebum → oleic acid 2) Why do we distinguish mycoses as inflammatory response invasive, subcutaneous, cutaneous… - Pityriasis versicolor (flaky, discoloured Invasive skin patches) - Severe fungal infections - Piedra (nodules on hair shaft) - Impact on internal organs/ deep tissue - Malassezia folliculitis (a fungal pimple) - Spread through the body – particularly in immunocompromised individuals. Common fungal causes o E.g., candidiasis and - Malassezia spp – a typical skin aspergillosis commensal - These are more life-threatening and - Trichophyton spp require prompt and aggressive tissue. Treatment – Subcutaneous Mycoses - good hygiene - Involves deeper layers of skin, - topical antifungal subcutaneous tissues, and sometimes - ketoconazole shampoo bones. - zinc pyrithione shampoo - Occur after traumatic implantation of - selenium sulphide lotion – cytostatic the fungus unto the skin. - E.g., Mycetoma Cutaneous mycoses – “ringworm” – tinea - Infections are chronic and cause Cutaneous mycoses are deeper than significant damage but rarely spread to superficial mycoses – more discomforting. organs - They are caused by dermatophytic fungi - Requires long-term treatment and – that eat skin keratin sometimes surgical intervention. - There are very high carriage rates of casual organisms – that are easily Cutaneous Mycoses passed via infected surfaces. - Affects the outer layers of skin, hair and - Usually treatable. nails. Disease names - Typically caused by dermatophytes – a - tinea corporis (ringworm of the body) group of fungi that feed off keratin - tinea capitis (ringworm on the scalp) - E.g., jock itch and athlete’s foot - tinea pedis (athletes’ foot) - Usually superficial and not life- - tinea cruris (ringworm on the groin) threatening – causes discomfort, cosmetic issues and secondary Common fungal causes bacterial infections if not treated. - Microsporum spp. - Trichophyton spp. Superficial Mycoses - Limited to the outer surface of skin and Clinical Aspects of Cutaneous Mycoses hair - Blister-like lesions – dry to leave a scaly - Infections of hair shafts known as ring Piedras - Without treatment may get hair loss, - Up to 20% of the population may be change of hair colour, and local infected inflammatory reactions. - Prevented by good personal hygiene - Can be highly adapted to humans or - Treated with cleansing agents transmitted from animals or via soil Treatment Summary of superficial, cutaneous and - Changing environment subcutaneous mycoses. - Topical antifungal agents such as o Miconazole o Terbinafine o Clotrimazole o Ketoconazole - It’s applied twice daily until symptoms resolve o Course is usually within one to two weeks (excluding nail infections) Invasive Fungal Infections (IFIs) Few fungi can cause IFI – they are mostly rare Subcutaneous Mycoses or geographically confined. - Deep tissue The big three of global importance are: - Caused by numerous soil fungi – especially black moulds (introduced via puncture wounds – and not spread from person to person) - Disease develops slowly with fungi spreading along lymphatic channels o Fungal tumour - Diagnosis: important as some infections mimic subcutaneous Aspergillus – aspergillosis mycoses. Primarily Aspergillus fumigatus (85%) and - Treatment: generally difficult - some Aspergillus flavus (5%) and other Aspergillus respond to anti-fungal and some may spp (5%). require surgical excision. - Acquired from the environment as Aspergillus makes millions of tiny spores. Aspergillosis is an opportunistic infection – that gets more serious with the level of immunocompromise in the host. - Aspergilloma (fungal ball) (TB) - Invasive pulmonary aspergillosis o Similar to bacterial pneumonia - Disseminated aspergillosis o Crossing blood-brain barrier. IFIs – candidiasis Candida Albicans and other Candida species cause candidiasis. - Single-cell yeast often forms pseudo hyphae This is a common cause of bloodstream infections in hospitals. Many Candida spp are commensal so: - Can be acquired endogenously or - Directly by person-to-person contact Candidiasis takes many forms 1) Candidiasis of the vagina - Vulvocandidiasis and Vulvovaginitis People most at risk are patients with severe 2) Candidiasis of the mouth immune defects. - Oral thrush or candidiasis Concept check 3) Candidiasis of the skin 1) What is meant by endogenous - Fingers (paronychia) and nails infection? (onychomycosis) It is an infection that originates from within the Disseminated candidiasis following immune body. It occurs when normal harmless micro- suppression, surgery, severe burns, and drug organisms change in the environment and then abuse. compromise the host. Cryptococcosis E.g., Candidiasis is an example of an Caused by cryptococcus species in two endogenous infection. This is because candida medically important species complexes. species are present in the mouth, gut and - Cryptococcus neoformans species vagina. Infection is caused when the immune complex system is weakened or when normal bacteria - Cryptococcus Gati species complex balance is disrupted. Grow as a yeast with a large capsule which is an important virulence factor Diagnosis and treatment of fungal infections - Thick polysaccharide coat (chitosan) Early, correct and proper treatment are - No longer virulent if removed essential. - Fungi are not harmed by antibacterials Cryptococcosis or antivirals - Cryptococcal pneumonia - Diagnosis done by culture and - Cryptococcal meningitis microscopy Process of cryptococcal infection Today immunological and molecular - Acquired from the environment (tree techniques are frequently used to speed up hollows, soil, guano) diagnosis – e.g., antigen dip–sticks, PCR with - The first site of infection is the lungs fungus–specific primers, MALDI – TOF (time of - From there it can disseminate to other flight mass spectroscopy) parts of the body – particularly the brain – lipid rich. Treatment with antifungal drugs The Azoles group is the most effective, There are a few effective anti–fungal drugs however, it does not target the cell wall just the which are grouped into 4 categories. cell membrane. 1) Nucleic acid synthesis inhibitors (5- flucytosine) 3) Polyenes 2) Ergosterol biosynthesis inhibitors This drug binds to ergosterol in fungal cell (azoles, terbinafine) membranes, causing cell membrane disruption 3) Polyenes (Nystatin – fungicidal) which leads to cell death. 4) Echinocandins (E.g., Caspofungin) - E.g., Amphotericin B and Nystatin This is also very cytotoxic – often referred to as Most antifungals target the cell wall or “amphoterrible” due to the severe side effects. membrane 4) Echinocandins This drug inhibits the synthesis of B-glucan which is an essential component of the fungal cell wall. - E.g., Micafungin and Caspofungin This targets the cell wall specifically (the beta- glucan synthesis in the cell wall) Summary of medically important fungi Problems with antifungal drugs - Fungi can cause a variety of clinical issues – ranging from hypersensitivity and toxicoses to severe, invasive infections. - Fungal infections are usually not severe but are highly prevalent. - Severity generally increases in immunocompromised hosts - They are difficult to treat – there are a few good targets, and all current antifungals have limitations. Concept check - Superficial and cutaneous infections 1) Nucleic Acid Synthesis Inhibitors are easier to treat as topical application This drug interferes with the synthesis of DNA has less toxic side – effects. and RNA in fungi. - E.g., Flucytosine Sterilisation Sterilisation is the complete destruction or It’s the most cytotoxic – as they interfere with removal of all living micro – organisms and their nucleic acid synthesis and hence it may affect spores from a product human cells as well. Preservation 2) Ergosterol Biosynthesis Inhibitors Prevent the growth of micro – organisms inside This drug inhibit the production of ergosterol a product using anti – microbial chemical which is a key component of fungal cell agents membranes. - E.g., Azoles (fluconazole) and Disinfection Allylamines (terbinafine) Reduction of the microbial population to some acceptable lower level for a defined purpose. What should be sterile? Any product that will be in contact with internal organs, broken skin and some mucosal surfaces (inside of the eye; urinary tract) - Parenteral products and their containers - Parenteral drug delivery devices - Ophthalmic preparations - Non – injectable sterile fluid - Surgical tools/device Two approaches to prepare sterile products Terminally sterilised product D – Value - Non – sterile product is placed in non – Time (or dosage) at a particular temperature (or sterile container, then sealed and intensity) that is necessary to reduce the viable ensemble is sterilised microbial population by 90% (or 1 log reduction) Aseptic Preparation - Components of product are sterilised If the bioburden of a product is 106 separately, then filled in a sterile microorganisms, what is the time required to container under aseptic techniques. reduce the number of microorganisms to 100 in a steam sterilisation at 121 degrees? Sterilisation mechanisms Given D121 = 1.5 min? The sterilisation process can remove or kill micro – organisms Answer = 4 x 1.5 = 6 min. - Physically - Chemically The choice of sterilisation method depends on - The ability of the product and its container to withstand the sterilisation conditions - Type of micro-organisms - Bioburden: total number of viable micro – organisms present in a product (before sterilisation) Special Curve The number of surviving microorganisms Z – Value plotted versus the time of exposure (or dose) of Increase in temperature to reduce the D – Value a killing process. of a microorganism by 90% (1 log reduction) - Linear relationship (most common) - The lower killing rate at high If in heat sterilisation reduces the number of microorganism number microorganisms by 90%, how long is needed to - Lower killing rate at lo survivor level. obtain the same microbial reduction if the temperature increases to 131? Given that D121 = 14 and Z = 10 degrees. Answer 1) 131 – 121 = 10 2) LogD131 divided by LogD121= 0.1 3) So, 0.1 x D121 = 1.4 minutes.  IF = 10t/d Question A batch of ampules of 2mL water for injection has a bacterial count of 50/ml. Give D121 = 1.5min (D value at 121 degrees) calculate: 1) The bioburden in each ampoule 2) The time required for batch sterilisation (SAL of 1 contaminated product in 106). 3) The time required to sterilise the ampoules at a sterilisation of 112 degrees, given a z value of 9. Sterile Answer A product is sterile when there is no surviving microorganisms. A sterilisation procedure cannot guarantee sterility, and can only express the likelihood of a product being free of microorganisms. In prac 1, e.g., the aseptic process includes transfer of liquids, dilution of liquids and dissolving of solids and capping containers. Different methods of sterilisation Sterility assurance (or microbial safety Heat sterilisation index) - Moist heat e.g., autoclave This is the probability of a single surviving - Dry heat e.g., oven organism that remains in a product. Gaseous sterilisation The acceptable sterility assurance level (SAL) - Ethylene oxide for parenteral products – 1 contaminated - Formaldehyde product in 1,000,000 (million) units or the probability of 99.9999…% that there is no Radiation sterilisation contaminated product. - Ionising radiation (gamma, electron) - Non – ionising radiation (UV) Filtration sterilisation - Depth filter - Membrane filter Moist (or steam) heat sterilisation Inactivation factor (IF) - Carried out in an autoclave Initial bioburden divided by the microbial count - Pressure is employed to raise the after sterilisation. temperature of stram above 100 degrees. E.g., - if the bioburden is 108 and the count of - Wet saturated steam – steam containing the microorganism after the sterilisation cycle water droplets, at saturation is 100 then IF = 106 temperature. - Dry saturated steam; steam not - IF is associated with the exposure time containing water droplets at saturation (t) and D – value as shown in the temperature. following equation.  - Autoclave is most effective using dry What cannot be sterilised by steam? saturated steam - Non – aqueous liquids (e.g., oily - When dry saturated steam comes to preparation) contact with a cooler surface, this - Powders causes water condensation and - In general, moist and heat-sensitive immediate release of the latent heat. products Why use dry saturated steam Types of autoclaves Dry saturated steam - Non – jacketed bench – top autoclave - Water condensation on bacteria - Downward (or gravity) displacement - Immediate release of latent heat autoclave - Hydration of bacteria and fast heat - Porous load (or vacuum) autoclave transfer to bacteria - Proteins on the surface of bacteria are Non – Jacketed bench top autoclave denatured It is like a large pressure cooker with water - Dry saturated steam has a better boiling at a temperature higher than its normal penetration into the pores of an item, boiling point. because of contacts in volume caused - Water in the base of the autoclave by condensation. - Steam removes the inside air - The pressure will rise until the required Superheated steam levels is reached. - Water steam does not condense - Steam acts like a hot dry gas with slow heat transfer - Superheated steam does not contract in volume. Standards for steam sterilisation Steam sterilisation stages - Heating: heat – up, air removal, and steam admission - Holding; exposing the load to steam - Cooling; dry and cool down If possible, a higher temperature (shorter cycle) Downward (or gravity) displacement should be chosen, to achieve a greater autoclave inactivation factor. The steam is prepared outside the chamber. Steam displaces air in the chamber by gravity without mechanical assistance, through a drain port. The chamber is surrounded by a steam – Sterilisation by steam jacket. The following can be sterilised by steam - Dressings - Surgical and diagnostic tools and equipment - Containers and packages - Aqueous solutions; injection, ophthalmic preparations, irrigation fluid  - Requires higher temperature and longer time than a moist sterilisation. Dry heat sterilisation When steam sterilisation is not effective - Steam cannot penetrate into the material - Products are steam sensitive (e.g., powder, wet granulation) - Glassware Gaseous Sterilisation Ethylene oxide C2H4O) and formaldehyde (CH2O). Its used for sterilising certain heat – sensitive articles (e.g., medical devices). Porous load (ore pre-vacuum) autoclave Air is mechanically removed from the chamber. Mechanism of action This allows the steam to penetrate porous - Alkylation of sulfhydryl (SH-) areas of the load that couldn’t otherwise be - Amino (NH2-) reached with simple gravity displacement. - Hydroxyl (OH-) - Carboxyl (COOH-) Vacuum system; faster and better steam - Groups of proteins and nucleic acids penetration into the pores of the load. - Alkylation; substitution of H by an alkyl group More sophisticated control systems to operate the sterilisation to operate the sterilisation cycles faster and more effectively. Ethylene Oxide (EO) - Colourless with uncharacteristic odour - Acute toxicity - Highly flammable and potentially explosive (normally diluted with CO2 to reduce the explosive hazard) - Readily absorbed by fabrics and plastic - Gas at room temperature, so should not be used in the open environment Advantages - Broad spectrum Dry heat sterilisation - Can sterilise at room or low Moist heat temperatures - Destroys the micro-organisms (or their - High penetration ability into materials spores) by hydrolytic damage - In the presence of water, the enzymes Disadvantages and proteins denature at lower - Expensive and long procedure temperatures. (compared to heat sterilisation) - Very hazardous gas and difficult to use Dry heat - Ethylene oxide absorbed on the treated - Less effective than moist sterilisation articles – the difficulty of gas removal Formaldehyde Gas - Colourless gas with an unpleasant Electromagnetic Spectrum odour - There is no abrupt change in going from - Neither explosive nor flammable one region to the next. - Acute toxicity and carcinogenic - Ultra – violet and gamma rays represent - Absorbed by fabrics and plastic (less fractions of the whole spectrum than EO) - High affinity to water - Can be polymerized at a temperature Ionising radiation below 80 degrees Types of radiation - Gamma irradiation Advantages - Electron irradiation - Less hazardous - Can be prepared in the form of a Gamma beams penetrate deeply but require solution. hours to sterilise large volumes. - Decontaminating room – heating paraformaldehyde at high humidity Electron beams have much lower penetrating (65%) power but usually require only a few seconds of exposure. Radiation Sterilisation - Ionising radiation (gamma, electron) Application and Mechanism - Non–ionising radiation (UV) Used For - Ionising radiation has a lower - Cold sterilisation method (room wavelength (1nm): and greater energy temperature) and penetration than non–ionising - Articles in the dried state (e.g., surgical radiation (UV – hundreds nm) instrument, powder) - Disposable items (e.g., plastic syringe) – large-scale sterilisation is possible in gamma radiation - Some pharmaceutical products Modes of action - Cleaving bonds in microbial DNA - Formation of free radical (OH- or O2-); The energy state of atoms damage DNA The levels are labelled by an integer n, called a - Increased effectiveness in the presence quantum number. of water, oxygen and elevated - The lowest energy state is called the temperature. ground state. All other states are called excited states Gamma radiation - Beyond an energy called the ionisation Emission from radioactive isotope cobalt – 60. potential, the single electron of a - The isotope is held as pellets packed in hydrogen atom is no longer bound to the steel rods. atom - The source is housed within a reinforced - When an atom change states it either concrete building and is sunk into a emits and absorbs photons with exactly water–filled tank. the amount of energy from one state to - Articles being sterilised are passed the other. through the irradiation chamber. Gamma radiation facility - Possible alterations and damage to A high-intensity gamma–ray emitter with a certain products and containers relatively long. - At high energies risk of X-ray induction in - The half-life is 5.26 years the product - The radiation is reduced by 10% each year Non–ionising radiation - Exposure time is 20 hours. Mostly used to sterilise the surface in a laminar flow hood. - Cannot sterilise solid materials as it has Advantages poor penetration - Suitable for heat and moist sensitive - UV light may damage the eyes and materials prolonged exposure can cause burns - Large volume can be sterilised in one and skin cancer load – good penetration power - Sterilisation can be carried out at room temperature Disadvantages - Requires highly specialised personnel equipment - Requires extensive precautions and expensive equipment - Suitable for large scale only – long sterilisation process - Possible alteration and damage to certain products and containers. Electron accelerators Spectra of sun rays - Electron beam is generated from a hot filament and accelerated by the high potential difference - Electron with max energy of 5 mev Microwave linear accelerator - Electron beam is generated from a hot filament and accelerated by synchronised microwave - Electron with max energy of 10 mev/ Advantages Filtration sterilisation - Suitable for heat and moist sensitive Removes microorganisms from liquids or gases materials with dual purposes. - Fast sterilisation process (in order of few - Sterilisation minutes) - Removal of non – vital particles - Sterilisation can be carried out at room temperature Mechanisms of action - Sieving; using a structure with pore sizes Disadvantages smaller than microorganism sizes - Suitable for products in small package – - Trapping; using a matrix of fine and low penetration power than gamma dense fibres to trap microorganisms - Requires extensive precautions and - Adsorption using a matrix in which expensive equipment microorganisms are adsorbed Factors contributing to the particle removal Membrane filter vs depth filter - Properties of liquid/gas and the velocity - Pore properties, texture od dilter matrix - Adsorption Filtration vs other sterilisation methods Advantages - Suitable for heat – sensitive products - Quick for low scale volume - In general, does not alter the properties of the products Disadvantages - Slow for large scale volume Membrane filter manufacturing - Cannot sterilise solid materials Gelling and Casting - It is an aseptic process - Polymers such as cellulose nitrate, - Risk of adsorption of product cellulose acetate, nylon etc.. components onto the filter materials - The polymer is mixed with a solvent to form a gel Membrane filter and depth filter - The solvent is evaporated to from a Membrane filter highly porous structure - Thin sheets of polymers (e.g., cellulose nitrate) with ultra-fine pores Irradiation – etch - Commonly used for sterilisation of - A thin film (5 – 10mm) of polycarbonate pharmaceutical liquids or polyester is exposed to a stream of - Pore size of 0.2um for removing bacteria charged particles and fungi - The passage of charged particles through the material causes fission in Depth filter molecules - Consists of thick layers of materials - The fission track made through the film such as porous ceramic or fibres of is etched out into a round, randomly polymers dispersed cylindrical pores. - Trapping or adsorption mechanisms - Pore density and size are controlled by - Is also used to remove big particles the duration of irradiation and etching before membrane filtration processes. Deep filter manufacturing Filtration process Fibrous pad filters - Carried out under positive or negative - Borosilicate, paper, nylon, polyester, pressure (syringe or pump_ cellulose acetate and woven wool fibre - The liquid can be collected into a final - In from of flexible pads – placed in a sterile container support ot holder - Filtration should be carried out under - Main problem – shedding fibres aseptic conditions. Sintered or fritted ware - Small particles of glass (or metal) which are heated to fuse together and from a porous structure. Selecting a suitable sterilisation method Monitoring the sterilisation process: monitoring the treatment that the product has undergone. - The assumption is that if the sterilising equipment is in working order, if the product has been subjected to a validated sterilising treatment and if good manufacturing practices (GMPs) prevail, then the batch will be sterile. The process 1) Equipment function tests o Heat distribution, temperature, Sterility assurance pressure, time, etc… - Sterility; absence of all viable 2) Exposure verification tests microorganisms in a medical and o Chemical indicators; physical pharmaceutical sense and/or chemical nature of the - sterility assurance; the probability of a indicator change after the single surviving organism remaining to sterilisation process contaminate a product o Biological indicators (BIs); the - acceptable sterility assurance level product is inoculated with a (SAL) for parenteral products; 1 known bacteria suspension. contaminated item in 1,000,000 or o After the sterilisation process, probability of 99.9999…% that there is the Bis are cultured. If no no contaminated item microbial growth takes place, the sterilisation has taken place. Two approaches to comply with sterility 3) Process validation – this can ensure the assurance desired sterile assurance only if it is Sterility testing: random microbiological reproducible. Therefore, during the assessment on a number of final products. sterilisation process o The bioburden should not change significantly o The containers and/or packaging should remain unchanged o The sterilisation protocol should be absolutely kept constant. Good Manufacturing Practices (GMP) Ensures that the products are consistently Example produced and controlled to the standard of quality. This may include: - Manufacturing processes; validated, clearly defined and systematically reviewed - Appropriately qualified and trained personnel - Adequate premises and spaces - Suitable equipment and services - Correct raw material, labelling and containers  - Suitable storage and transport Comparison with other antimicrobials - Appropriate recording and reporting Chemical antimicrobial agents (disinfectants, procedures. antiseptics, preservatives) are NOT selective to a certain type of microorganism. Chemical Antimicrobial Agents - Different from anti- Disinfection bacterial/fungal/viral/protozoal/parasiti Antimicrobial agents are applied to the surface c agents which are selective. of non–living objects to destroy - Antibiotics are a subset of antibacterial microorganisms. agents that are natural biological - To clean surfaces in the dental surgery structures. and agents used for cleaning - Antibacterial agents also include semi instruments or fully synthetic - Reduction of the microbial population to acceptable lower levels for a defined Antimicrobial agent mechanism purpose. o Low levels; kill most vegetative bacteria, fungi, and viruses but does not include some of the more resistant micro-organisms o Intermediate level: kills all vegetative bacteria and most fungi and viruses. o High level: kills all microorganisms but not Factors influencing antimicrobial agents necessarily bacterial spores. Concentration of antimicrobial agent - Higher the concentration the more Antisepsis effective Destruction or inhibition of micro-organisms on living tissues. Temperature - Adequate antimicrobial activity but not - Bactericidal activity is increased at toxic or irritating to tissue. higher temperature - Used before surgery or on a wound to - Increasing the temperature is not help prevent the spread of infections. always applicable - Mouthwashes, root canal treatment o Degradation of antimicrobial medicaments, hand hygiene (isopropyl agents alcohol wipe) o Other ingredients may be heat – sensitive Preservation - Added in pharmaceutical preparations Acidity to prevent microbial growth and - For the anti-microbial molecule, spoilage changing pH may change the degree of - In non–sterile products, ionisation microorganisms may be introduced - For the cells, changing pH may change during manufacturing the negative charges of bacterial cell - Minimising the risk of contamination in wals. sterile products due to administration - Commonly used preservatives in topical and transdermal products Interfering substances Different types of bacteria Organic matter, such as serum, blood, pus, Mycobacteria earth, food residues, milk, skin, fats, may - Smallest bacteria decrease the effect. - Low cellular permeability - Surfactants, such as soap detergent - Have a very thin cell wall (the o May increase the effect by peptidoglycan layer is thin) and the cell increasing bacterial cell wall consists of 60% of a hydrophobic permeability waxy lipid o May decrease the effect by interacting with the antimicrobial Gram Positive and Negative agent - Gram+ bacteria have a thicker peptidoglycan cell wall Microorganism - Gram- bacteria have a thinner - Formation of biofilms peptidoglycan and an additional - Natural resistance of microorganisms lipopolysaccharide layer. - Microbial resistance due to o Lipopolysaccharide provides density/growth conditions. additional protection. So gram- negative bacteria are more resistant to antimicrobial agents Natural resistance of microorganisms Proteinaceous infectious (prion) particles Microbial resistance due to density and made only of protein (lack of DNA). growth condition - WHO recommends the combined use of - The larger the number of NaOH and autoclaving at 134 degrees microorganisms (greater bioburden), the longer it takes to kill them - Bacteria in the logarithmic phase are more susceptible to antimicrobial agents than bacteria in the stationary phase Different phases of bacteria - Spores have a low water content - Spores are covered with tough materials - Some spores can resist boiling water and ethanol Biofilms A community of microorganisms attached to a surface. It may consist of multiple bacterial species. Biofilm causes severe medical problems Aldehydes - Infection of implantable and indwelling E.g., Formaldehyde (CH2O) medical devices, biocorrosion of - Usage; disinfection and sterilisation materials, tooth caries - Mechanisms of action - Bacteria as biofilms are several hundred o Action on cell wall – covalently times more resistant to microbial agents binding to cell wall than free cells o Act on cytoplasm – denature and - In nature, most bacteria aggregate as coagulate proteins biofilms - Advantages: rapid rate of kill, highly effective disinfectant, organic matter Formation of biofilms has little effect - Conditioning layer; deposition of - Disadvantages; irritates, toxic organic molecules (proteins) carcinogenic - Bacteria will strike and adsorb to the surface for some time and then desorb Biguanides - Attached bacteria excrete extracellular E.g., Chlorhexidine matrices (sticky polymers) - Usage: topical antiseptic, contact lens - The biofilms are permeated at all levels solution, bladder irrigation, oral by a network of channels antiseptic in dental application - Biofilms spread by releasing new - Advantages: Non–toxic to mucous pioneer bacteria to colonise membranes and skin downstream section. - Disadvantages: low solubility in water, low antimicrobial effect Families of chemical antimicrobial agents - Recall: chlorhexidine as a preservative - Alcohols for Cetomacrogol lotion - Aldehydes - Biguanides Phenolic compounds - Phenolic compounds E.g., thymol and chlorocresol - Halogens - Usage: disinfectants, preservatives, - Organic and inorganic acids antiseptics - Heavy metals derivatives - Mechanism of action - Peroxygens o Act on cell membrane - Surfactants - Advantages o Persistent Alcohol o Good bactericidal activity E.g., Ethanol and isopropanol. - Disadvantages o Poor aqueous solubility - Usage: disinfectant, antiseptic, o Caustic effect on skin and tissue preservative (e.g., benzyl alcohol). o Not to be used where they can - Mechanism: act on the cell membrane, contaminate the food resulting in a leakage of cell o Activity is reduced in the constituents presence of organic matters - Applications: 70% (below 50% ethanol is not effective) - Advantage: fast-acting, compatibility, acts and evaporates, topical application - Disadvantage: flammable/ poor penetration in organic matters Bisphenols; are composed of two phenolic Organic and inorganic acids groups E.g., acetic acid (vinegar), benzoic acid (occurs E.g., triclosan naturally in many plants), salicylic acid - Phisohex (cosmetics), lactic acid (an ingredient in sweat) - Application: can be used as an - Usage antiseptic and preservative in soap, o Widely used for preservation of toothpaste, shower foams, deodorants food and pharmaceutical and fabrics products; anti-fungal topical - Lower toxicity and irritancy than mono- treatment; anti-acne phenolic compounds have low water - Mechanism of action solubility o On cell membrane; inhibition of - In 2017, the FDA rules; that companies energy processes in cell would not be able to triclosan or 23 - Advantages other active ingredients in these o Low toxicity – ideal for food and products without premarket review due cosmetics to insufficient data regarding their safety - Disadvantages and effectiveness. o Low bactericidal activity o More toxicity at a lower pH value Halogens o Inactivated at a higher pH value - Chlorine compounds (e.g., sodium hypochlorite NaClO in Milton solutions). Heavy metal derivatives - Mechanism of action E.g., silver, mercury, copper o Act on cell membrane: - Usage halogenation of amino acids in o Mercury; prevent mould in proteins painting o In cytoplasm; inhibition of DNA o Copper; kill green algae in water synthesis reservoirs - Usage o Silver; antiseptics and o Disinfection of water supplies, preservatives in pharmaceutical surfaces, equipment products o Preservation of food - Dental amalgam (silver filling) is a mixture of mercury, silver, copper, tin Iodine Compounds and zinc used to fill cavities in teeth, - Betadine Antiseptic oral rinse being gradually replaced by resin – - In comparison to chlorine compounds based composites o Iodine has lower solubility and - Advantages less irritant o Biocidal at low concentrations o Iodine is less susceptible to - Disadvantages inactivation by organics o Corrosive/toxic to living cell o Iodine can stain skin and is less effective against spores Peroxygens - Povidone–iodine is designed using a E.g., hydrogen peroxide similar approach to that of making - Usage polymerized quaternary ammonium o Disinfectant of soft contact compounds. lenses o General disinfectant and antiseptic - Mechanism of action o Oxidise thiol groups in enzyme and proteins o Generate free hydroxyl radical (OH-) to damage DNA Advantages Preservation of medicine and cosmetics - High-level bactericidal activity Non–sterile product: to prevent the spoilage - Environmentally friendly and degradation of product due to metabolic (biodegradable) activity of microorganisms. - At high concentrations/temperatures kill bacterial spores The rate of deterioration depends on - Type and quantity of microorganisms Disadvantages - Physiochemical properties of - Corrosive, irritant at high concentrations environment - Unstable, so benzoic acid is added as a - Molecular structure of chemicals in the stabilizer product Surfactants Sterile products: to prevent microbial - Structure; have a hydrophobic group contamination during usage. The rate of in–use and a hydrophilic group contamination depends on: - Mechanism of action - Type of the container o Breaks the oily film into tiny - Operator – introduced contamination droplets oil o Microbes are removed Different techniques of preservation mechanically by scrubbing and Chemical preservatives washed out with water - Adding antimicrobial agents into the - Two main types of surfactants product formulation o Cationic: hydrophilic group ionise to cation Storage temperature o Anionic; hydrophilic group ionise - Short-term storage: cool place (8 – 12 to anion. degrees) Quaternary ammonium compounds - Long-term storage: freeze (below -20 E.g., benzethonium chloride degrees) - Usage o Food processing industry Extreme pH prevents bacterial spoilage. o Antiseptic (skin, mucous membranes Moisture content o Throat infection) - Decrease the available water o Preservatives (eye drops) - Avoid surface water condensation - Mechanism of Action o On the cell cytoplasmic Package design membrane, change the cell - Type of materials/moisture-proof permeability and cause the - Good sealing, narrow nozzles, screw- leakage of cytoplasmic capped tubes constituent - Advantages Oxygen level: aerobic organisms need oxygen o Surface active – cleaning to survive. property o Non–toxic, stable at storage, no odour - Disadvantages o Low antimicrobial activity o Inactivated chemical agents