Institute for Microbiology, Medical Faculty of Masaryk University and St. Anna Faculty Hospital in Brno Miroslav Votava RESISTANCE OF MICROBES TO THEIR ENVIRONMENT (TENACITY) The 4th lecture for 2nd-year students of General Medicine March 9th, 2015 Division of bacterial cell – revision capsule cytoplasmatic membrane bacterial cell wall fimbriae nucleoid ribosomes plasmids granules vacuole flagellum septum Div. & arrrangem. of cocci – revision •Cocci, dividing in one plane: streptococci chains • •Cocci, in different planes: staphylococci clumps • • •Cocci, in two perpendicular planes: • micrococci tetrads • Division and arrangement of rods – revision •Rods, transverse division: majority (chains of • rods) • • •Rods, lengthwise division: mycobacteria • corynebacteria • (arrangement in palisades) Generation time – revision •Generation time = duration of the growth cycle = = duplication time = duration of doubling the number of bacteria •Generation time of bacteria: on average cca 30 min • Mycobacterium tuberculosis • approximately 12 hrs • •Since during each generation time the number of bacteria doubles, bacteria multiply by geometric progression Geometric progression – revision •If the generation time is 30 min, after 24 hrs theoretically one cell gives origin to 248 = 2.8×1014 cells, actually it is by approximately 5 orders less (i.e. around 109 cells) • •109 bacteria is such an amount that it is visible even by the naked eye: •Liquid medium (broth) becomes 1. cloudy or 2. a sediment appears at the bottom or 3. a pellicle is seen at the top •On a solid medium (agar) a bacterial colony forms • • What is a bacterial colony – revision •Bacterial colony = a form on the surface of the agar, containing mutually touching cells, cca 109 living and cca 105 already dead • •Appearance of the colony depends apart from other things on the • microbial species (e.g. on the size of its cells) • sort of culture medium (e.g. on the amount of its nutrients) • distance among colonies (the higher distance, the larger and more typical the colony) • •By appearances of the colonies microbiologists recognize different microbes • Features of a bacterial colony – revision •Bacterial colony can have up to 10 features: • 1. Size – usually around 1-2 mm • 2. Shape – round, oval, irregular, lobular etc. • 3. Profile – flat, convex, dish-shaped etc. • 4. Margins – straight, fibrous, with projections etc. • 5. Surface – smooth & glossy, matt, rough, wrinkled • 6. Transparency – transparent, nontransparent • 7. Colour – colourless, pigmented (yellowish etc.) • 8. Changes in vicinity – pigmentation, haemolysis • 9. Consistency – sticky, mucous, crumbly, rooted •10. Smell – foul, pungent, of jasmin, sperm, fruit etc. • Microbial growth curve – revision 10 8 6 4 2 lag phase stationary phase approximately 24 hrs time Factors of the outer environment •water •nutrients •temperature •osmotic pressure •pH •redox potential •radiation •toxic substances Water shortage •Water = 80 % live weight of the bacterial cell • (only 15 % live weight of the bacterial spore) • •Hygrophile organisms (most of bacteria) need freely accessible water •For xerophiles (actinomycetes, nocardiae, moulds) water bound to the surface of environmental particles (e.g. in soil) suffices Water availability •Degree of water availability = water activity of the environment (aw) •aw of pure water = 1.0 •aw is inversely related to osmotic pressure (the higher the osmotic pressure, the lower aw) • •The water activity (aw) tolerated by different microbes: •G– bacteria aw ≥ 0.95 (meat) •G+ bacteria and most yeasts aw ≥ 0.9 (ham) •staphylococci aw ≥ 0.85 (salami) •moulds and some yeasts aw ≥ 0.6 (chocolate, • honey) Resistance to drying up •Very sensitive: agents of STD – gonococci, • treponemes •Less sensitive: all Gram-negative bacteria •A bit more resistant: skin flora – staphylococci, • corynebacteria • acidoresistant rods – • mycobacteria •Rather resistant: xerophiles – actinomycetes, • nocardiae, moulds • parasite cysts, helminth eggs •Highly resistant: bacterial spores • Practical application of water shortage •Lowering water activity stops action of most microbes → we use it for food preservation • •drying – meat, mushroom, fruit (prunes) •concentration – plum jam •salting – meat, fish, butter •sugaring – sirups, jams, candied fruit • Nutrient deficiency •Microorganisms do not multiply in clean water •The problem lies in keeping water pure •After some time, even in distilled water e.g. Pseudomonas aeruginosa or Pseudomonas fluorescens start to multiply •In shower sprinklers: Legionella pneumophila grows (and can cause pneumonia) •However, Salmonella Typhi lives longer in well water than in waste water – why? • Temperature •Cardinal growth temperatures: •Minimum – sometimes <0 °C (in sea water) •Optimum – psychrophiles: 0 – 20 °C • mesophiles: 20 – 45 °C (medically • important microbes) • thermophiles: 45 – 80 °C • hyperthermophiles: >80 °C •Maximum – sometimes >110 °C (in geysers) •Growth temperature range: • narrow (gonococci 30 – 38.5 °C) • wide (salmonellae 8 – 42 °C) The influence of cold •Cold shock: gonococci will die if inoculated at cold agar media freshly taken out of the fridge •Growth temperature minimum: •at 5 °C: salmonellae & campylobacters survive, yersiniae & listeriae even multiply! •Lyophilization, used for the conservation of microbial cultures ´ common freezing! •Slow freezing and repeated defrosting is somewhat harmful, but most microbes survive it •Tissue cysts of Toxoplasma gondii in meat do not survive common freezing The influence of heat •The temperature higher than optimum → heat shock and gradual dying of cells • •The number of killed cells depends on the duration of the exposure to higher temperature • •The relation between the number of surviving cells and the duration of heating is logarithmic one • •The time needed for exterminating (killing) the whole population depends on its size (on the initial number of microbes) Temperature – important parameters I •The relation between the duration of heating and the number of surviving microbes • • •Log10 number •of survivors • 6 D = decimal reduction time = • 5 = the time required to reduce • 4 the No of microbes to 1/10 = • 3 = the time required to kill 90 % of • 2 D microbes present (at the • 1 specific temperature) • • • 1 2 3 4 5 6 (min) Temperature – important parameters II •Thermal death point (TDP) = the lowest temperature at which a microbial suspension is killed in a specific time (usually in 10 minutes) •TDP depends not only on the nature of the microbial species but also on its stage, number and on the local environment •Thermal death time (TDT) = the shortest time needed to kill all microbes in a suspension For most bacteria it averages 10-15 minutes at 60-65 °C Osmotic pressure •Hypotony – the damage is prevented by the cell wall •Hypertony mostly hinders microbes in multiplying (therefore fruit is candied, meat salted) •Higher osmotic pressure is endured by: •halophiles – halotolerant: enterococci (6.5% NaCl) • staphyloccoci (10% NaCl) • – obligate: halophilic vibria (in sea water) •moulds – tolerate higher content of saccharose (in jams) pH •Neutrophiles: growth optimum at pH 6 až 8 – most •Alkalophiles: e.g. Vibrio cholerae (pH 7.4-9.6) alkalotolerant: Proteus (it splits urea), Enterococcus (broad range of pH 4.8-11) •On the contrary, there are microbes sensitive to extremes of pH: e.g. gonococci •Acidophiles: facultative: yeasts, moulds, lactobacilli (>3), coxiellae (tolerate low pH of phagosome) obligate: Thiobacillus thiooxidans (pH <1) •Microbes sensitive to low pH: mainly vibrios, streptococci, putrefactive bacteria; low pH hinders most bacteria •Why sparkling water lasts longer? Because its pH is lower •Low pH keeps spores from germinating → botulism can be obtained from oil-preserved mushrooms or preserved strawberries, not from pickled gherkins or mixed pickles • • Redox potential (rH) •Level of rH depends both on the composition of the environment and of the atmosphere •Aerobes – need high rH levels (>200 mV) •Anaerobes – need low rH levels (≤0 mV) •Anaerobes are killed by O2, aerobes without O2 will live •Even so, anaerobes prosper both in nature and in our bodies – thanks to the cooperation with aerobes and facultative anaerobes (e.g. in biofilms) •Anaerobes in the body: • large intestine (99 % of bowel microorganisms) vagina oral cavity (sulci gingivales) • • • Radiation •UV radiation (maximum effect around 260 nm) •In nature airborne bacteria protect themselves by pigments → they have coloured colonies •Artificially: UV radiation is used for disinfection of surfaces, water, air; in PCR laboratories for destroying residues of DNA •Ionizing radiation (X and gamma radiation) •For sterilizing disposable syringes, infusion sets, materials for dressing and sewing, tissue grafts, some drugs, even waste and food (not in EU) •Record holders for radiation resistance: Deinococcus radiodurans and bacterial spores • Toxic substances •Their influence depends on the concentration and duration of exposure •Various microbes markedly differ in relative resistance to different types of toxic substances •In general (and contrary to drying): G– bacteria are more resistant to toxic substances than G+ bacteria (because of different structure of bacterial cell wall → presence of enzymes in periplasmatic space of G– bacteria) • •For application it is vital to know the effects of the particular substances used for disinfection • • Bacterial cell wall •G+ G– • lipoteichoic acid O-antigen lipopoly- • inner polysaccharide saccharide • lipid A (endotoxin) • murein • • • porin • • outer • membrane • lipoprotein • ENZYMES periplasmatic • space • • inner membrane • (G–) •cytoplasmatic membrane (G+) Sterilization versus disinfection •Sterilization = removal of all microorganisms from objects or environment • •Disinfection = removal of infectious agents from objects and environment or from the body surface •Disinfection aims at breaking the chain of infection transmission •Biocides = a new general term including also disinfectants Types of disinfectants 1.Oxidizing agents (peracetic acid, H2O2, O3) 2.Halogens (hypochlorite, sol. iodi) 3.Alkylating agents (aldehydes) 4.Cyclic compounds (cresol, chlorophenols) 5.Biguanides (chlorhexidine) 6.Strong acids and alkali (e.g. slaked lime) 7.Heavy metal compounds (Hg, Ag, Cu, Sn) 8.Alcohols (ethanol, propanols) 9.Surface active agents (QAS; e.g. cetrimid) 10. Others (e.g. crystal violet & other dyes) 11. • Relative resistance of different agents to biocides •Enveloped viruses herpesviruses •Some protozoa very susceptible Trichomonas •Gram-positive bacteria Streptococcus •Gram-negative bacteria Salmonella •Yeasts susceptible Candida •Moulds Trichophyton •Naked viruses enteroviruses •Protozoal cysts relatively resistant Giardia •Acidoresistant rods Mycobacterium •Helminth eggs Ascaris •Bacterial spores very resistant Clostridium •Coccidia Cryptosporidium •Prions extremely resistant agent of CJD • 1. • Universally effective biocides • • 1. •On small, naked viruses: oxidizing agents • halogens • aldehydes • strong acids and alkali •On mycobacteria: oxidizing agents • aldehydes • lysol • strong acids and alkali •On bacterial spores: (oxidizing agents) aldehydes strong acids and alkali • (not alcohols!) • Curriculum of lectures, 2014/15, spring term • 1. Microbiology and medicine • 2. Morphology and structure of bacteria • 3. Bacterial growth, growth curve • 4. Tenacity of microbes (their resistance to the environtment) • 5. Microbial biofilm • 6. – 7. Pathogenicity and virulence • 8. – 9. Pathogenesis of infection •10. – 11. Course and forms of infection •12. – 13. Active and passive immunization •14. – 15. Antimicrobial therapy • Recommended reading material •Paul de Kruif: Microbe Hunters •Paul de Kruif: Men against Death •Axel Munthe: The Story of San Michele •Sinclair Lewis: Arrowsmith • •Could you kindly supply me with another work in •connection with microbes or at least medicine? •Please mail me your suggestions at: •mvotava@med.muni.cz • •Thank you for your attention • •