Miroslav Votava Biofilms in Medicine The 5th lecture for the students of General Medicine March 16, 2015 Department of Microbiology, Masaryk University Medical School and St. Anna´s Faculty Hospital In Brno Water shortage – revision •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 Resistance to drying up – revision •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 – revision •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 – revision •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 – revision •Cardinal growth temperatures: •Optimum – psychrophiles: 0 – 20 °C • mesophiles: 20 – 45 °C (medically • important microbes) • thermophiles: 45 – 80 °C • hyperthermophiles: >80 °C • •Growth temperature range: • narrow (gonococci 30 – 38.5 °C) • wide (salmonellae 8 – 42 °C) The influence of cold – revision •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! •Tissue cysts of Toxoplasma gondii in meat do not survive common freezing The influence of heat – revision •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 killing the whole population depends on the initial number of microbes Thermal death time – revision •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 (important exception: spores) Osmotic pressure – revision •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 – revision •Neutrophiles: growth optimum at pH 6 až 8 – most microbes •Alkalophiles: e.g. Vibrio cholerae (pH 7.4-9.6) alkalotolerant: Proteus (it splits urea), Enterococcus (broad range of pH 4.8-11) •Microbes sensitive to extremes of pH: e.g. gonococci •Acidophiles: facultative: yeasts, moulds, lactobacilli (>3), coxiellae (tolerate low pH of phagosome) •Microbes sensitive to low pH: mainly vibrios, streptococci, putrefactive bacteria; low pH hinders most bacteria •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) – revision •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 – revision •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 •Ionizing radiation (X and gamma radiation) •For sterilizing disposable syringes, infusion sets, materials for dressing and sewing, tissue grafts, some drugs, waste and food (this not in EU) •Record holders for radiation resistance: bacterial spores • Toxic substances – revision •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 • • Sterilization versus disinfection – revision •Sterilization = removal of all microorganisms from objects or environment • •Disinfection = removal of infectious agents from objects and environment or from the body surface • •Biocides = a new general term including also disinfectants Types of disinfectants – revision 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 – revision •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!) • Two forms of microbial growth •Growth in the planctonic form: • Isolated microbial cells freely float • in the fluid environment • •Growth in the biofilm form: • Microbial cells have a natural tendency to • stick to each other and to solid surfaces and • to form a community interconnected with an • extracellular mass • • • Which form is more frequent? •Planctonic form •Common in the laboratory (e.g. in broth) • •Biofilm form •Primary in the natural environment because it is more advantageous for the microbes Definition of biofilm •Sessile microbial community •Its cells are irreversibly attached to a substratum or interface or to each other •They are embedded in a matrix of extracellular polymeric substances that they have produced •They exhibit an altered phenotype with respect to the growth rate and gene transcription •They are highly resistant to outer influences Dental plaque as a biofilm •Nowadays, dental plaque is viewed as a microbial biofilm •It is composed of numerous bacteria that –live in close juxtaposition –communicate mutually –influence each other •In disease, the balance of predominant bacterial populations typically shifts Architecture of dental biofilm •Original assumption: •Dental plaque has a compact structure •New technologies (confocal microscopy) say otherwise: •Dental plaque has an open architecture similar to other biofilms, with channels and voids Biofilm architecture skeno2 Microcolonies embedded in extracellular matrix form fungus-like structures interwowen with system of channels and voids Biofilm architecture Obraz Mutual relations between biofilm bacteria •Bacteria in plaque communicate mutually •through coaggregation and coadhesion (physically) •through conventional metabolic interactions (biochemically) •via small diffusible signalling molecules (quorum sensing) Coaggregation in plaque •E.g. anaerobic Fusobacterium nucleatum rod literally forms bridges between •early colonizers of the tooth surface – streptococci, actinomycetes – and •anaerobic late colonizers – Treponema denticola, Porphyromonas gingivalis, Tannerella forsythia (former Bacteroides forsythus) et al. • Quorum sensing in plaque •Quorum sensing: • During multiplication the individual cells emit chemical signals • (e.g. competence-stimulating peptide CSP in Streptococcus mutans) • After the cells reach certain number (quorum) • the higher concentration of signals triggers the following change in cell properties: • - shutting off some so far operating genes • - switching on other genes, resulting in • - production of new products (chiefly exopolysacharides; • in S. mutans it means – formation of biofilm • – induction of acid tolerance and • genetic competence) • Dental plaque – conclusion •Oral bacteria in plaque: •not independent entities •function as a microbial community –coordinated –spatially organized –fully metabolically integrated – •The properties of the community: •greater than the sum of the component species Biofilm in dental office – I •Dental unit waterlines = ideal environment for microbial colonization → biofilm •Source of microbes: –public water supply –occasionally aspirated patient saliva •Consequence of biofilm in dental waterlines: elevated concentrations of microorganisms in water emitting e.g. from high-speed handpiece – – Biofilm in dental office – II •Microbes found in dental unit water in significant concentrations: • pseudomonads (chiefly P. aeruginosa potentially pathogenic for susceptible host) • legionellae (some species are pathogenic, dental personnel has higher rates of Legionella antibodies) •Evidence suggests exposure of patients and dental staff to potential bacterial pathogens via dental water – – Biofilm in dental office – III •As yet, no efficient way of controlling dental water unit biofilm is known •Main interim recommendation: •Run water for several minutes at the beginning of each clinic day •Run high-speed handpieces for 30 seconds after use on each patient •Follow the instruction of the dental unit´s manufacturer Chronic infections of natural bodily surfaces dental caries (alpha streptococci) periodontitis (gramnegative oral anaerobes) otitis media (Haemophilus influenzae) osteomyelitis (Staphylococcus aureus) cholecystitis (enterobacteriae) prostatitis (Escherichia coli) subacute bacterial endocarditis (alpha streptococci) pneumonia during cystic fibrosis (Pseudom. aeruginosa) Chronic infections of artificial devices central venous catheters (coag. neg. staphylococci, candidae) artificial cardiac valves (Staph. aureus, Staph. epidermidis) artificial joints (Staphylococcus aureus, Staph. epidermidis) surgical sutures (Staph. aureus, Staph. epidermidis) vascular grafts (Gram-positive cocci) endotracheal cannulae (various bacteria and yeasts) intrauterine devices (Actinomyces israelii) urinary catheters (E. coli and others, mostly G-negative rods) contact lenses (Pseudomonas aeruginosa, G-positive cocci) Recommended reading material •Paul de Kruif: Microbe Hunters •Paul de Kruif: Men against Death •Axel Munthe: The Story of San Michele •Sinclair Lewis: Arrowsmith •André Maurois: La vie de Sir Alexander Fleming • • •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 • •