06 resistance of_microbes_to_their_environmen__tenacity_

InstituteInstitute for for Microbiology, Medical Faculty of Masaryk University Microbiology, Medical Faculty of Masaryk University

and St. Anna Faculty Hospitaland St. Anna Faculty Hospital in Brno in Brno

Miroslav Votava, Vladana WoznicováMiroslav Votava, Vladana Woznicová

RESISTANCE OF MICROBES TO RESISTANCE OF MICROBES TO THEIR ENVIRONMENTTHEIR ENVIRONMENT

(TENACITY)(TENACITY)

Lecture for 2nd-year studentsLecture for 2nd-year studentsMarch 2March 233, 200, 20099

Factors of the outer environmentFactors of the outer environment

• waterwater

• nutrientsnutrients

• temperaturetemperature

• osmotic pressureosmotic pressure

• pHpH

• redox potentialredox potential

• radiationradiation

• toxic substancestoxic substances

Water shortage Water shortage

Water – Water – 80 %80 % live weight of bacterial cell live weight of bacterial cell

(only 15 % live weight of bacterial spore)(only 15 % live weight of bacterial spore)

Hygrophile organismsHygrophile organisms (most of bacteria) (most of bacteria)

need freely accessible waterneed freely accessible water

For For xerophilesxerophiles (actinomycetes, no (actinomycetes, noccardiae, ardiae, moulds) water bound to surface of moulds) water bound to surface of environmental particles (e.g. in soil) sufficesenvironmental particles (e.g. in soil) suffices

Water availabilityWater availability

Degree of water availability =Degree of water availability = water activity of the water activity of the environment (aenvironment (aww))

aaww ofof pure water =pure water = 1,01,0aaww is inversely related to osmotic pressure (the higher is inversely related to osmotic pressure (the higher

the osmotic pressure, the lower athe osmotic pressure, the lower aww))

The degree of water activity (aThe degree of water activity (aww) tolerated by different ) tolerated by different microbes:microbes:

G- G- bacteriae bacteriae a aw w ≥ 0,95 (meat)≥ 0,95 (meat)G+ bacteriae and most yeasts G+ bacteriae and most yeasts aaw w ≥ 0,9 (ham)≥ 0,9 (ham)staphylococci staphylococci aaw w ≥ 0,85 (salami)≥ 0,85 (salami)moulds and some yeasts moulds and some yeasts aaw w ≥ 0,6 (chocolate, ≥ 0,6 (chocolate, honey)honey)

Resistance to drying upResistance to drying up

Sensitive:Sensitive: agents of STD – gonococci, agents of STD – gonococci,

treponemestreponemes

More resistant:More resistant: skin flora – staphylococci, skin flora – staphylococci,

corynebacteriacorynebacteria

acidoresistant rods (mycobacteria) acidoresistant rods (mycobacteria)

Resistant:Resistant: xerophiles – actinomycetes, xerophiles – actinomycetes,

nocardiae, moulds nocardiae, moulds

parasite cysts, helminth eggs parasite cysts, helminth eggs

Highly resistant:Highly resistant: bacterial spores bacterial spores

PracticalPractical application of water application of water shortageshortage

Lowering water activity stops action of most Lowering water activity stops action of most microbes microbes → we use it for→ we use it for foodfood preservationpreservation

• dryingdrying – meat, mushroom, fruit (prunes) – meat, mushroom, fruit (prunes)

• concentrationconcentration – – makingmaking plum jam plum jam

• saltingsalting – meat, fish, butter – meat, fish, butter

• sugaringsugaring – sirups, – sirups, jams, candied fruitjams, candied fruit

Nutrient deficiency Nutrient deficiency

Most microorganisms Most microorganisms dodo not not multiply multiply in clean in clean waterwater

The problem lies in The problem lies in keeping water purekeeping water pure

After some time, even in distilled water e.g. After some time, even in distilled water e.g. Pseudomonas aeruginosa Pseudomonas aeruginosa or or Pseudomonas Pseudomonas fluorescens fluorescens start to multiply start to multiply

In shower sprinklers: In shower sprinklers: Legionella pneumophilaLegionella pneumophila

TemperatureTemperature

Cardinal growth temperatures:Cardinal growth temperatures: Minimum – sometimes Minimum – sometimes <0 °C<0 °COptimum – psychrophiles: 0 – 20 °C Optimum – psychrophiles: 0 – 20 °C mesophiles: 20 – 45 °Cmesophiles: 20 – 45 °C thermophiles: thermophiles: 45 – 80 °C45 – 80 °C hyperthermophiles: >80 °Chyperthermophiles: >80 °CMaximum – sometimes Maximum – sometimes >110 °C>110 °C

Growth temperature range: Growth temperature range: narrow (gonococci 30 – 38,5 °C)narrow (gonococci 30 – 38,5 °C)wide (salmonellae 8 – 42 °C)wide (salmonellae 8 – 42 °C)

The influence of coldThe influence of cold

Cold shock:Cold shock: gonococci will die if inoculated at cold gonococci will die if inoculated at cold agar media freshly taken out of the fridge agar media freshly taken out of the fridge

Low growth temperature minimum:Low growth temperature minimum:

at 5 °C: salmonellae at 5 °C: salmonellae & campylobacters survive, & campylobacters survive, yersiniae & listeriae multiplyyersiniae & listeriae multiply

Common Common freezingfreezing lyophilization lyophilization

Slow freezing and repeated defrosting is somewhat Slow freezing and repeated defrosting is somewhat harmful, but most microbes survive itharmful, but most microbes survive it

Tissue cysts of Tissue cysts of Toxoplasma gondii Toxoplasma gondii in meat do not in meat do not survive common freezingsurvive common freezing

The influence of heatThe influence of heatTemperature higher than optimum Temperature higher than optimum → heat shock → heat shock

and and gradualgradual dying dying of cells of cells

Number of killed cells depends on the Number of killed cells depends on the duration of duration of the exposure the exposure to higher temperatureto higher temperature

Relation between the number of surviving cells and Relation between the number of surviving cells and the duration of heating is the duration of heating is logarithmic logarithmic oneone

Time needed to exterminating the whole population Time needed to exterminating the whole population depends on its size (on the depends on its size (on the initial number initial number of of microbes)microbes)

Temperature – important Temperature – important parameters Iparameters I

The relation between the duration of heating The relation between the duration of heating and the number of surviving microbes and the number of surviving microbes

LogLog1010 number number of survivors of survivors 6 6 D =D = decimal reduction timedecimal reduction time == 5 5 = the time required to reduce= the time required to reduce 4 4 the No of microbes to 1/10 =the No of microbes to 1/10 = 3 3 = the time required to kill 90 % of= the time required to kill 90 % of 2 2 DD microbes present (at themicrobes present (at the 1 1 specific temperature)specific temperature)

1 2 3 4 5 6 (min)1 2 3 4 5 6 (min)

Temperature – important Temperature – important parameters IIparameters II

Thermal death pointThermal death point (TDP) = the lowest (TDP) = the lowest temperature at which a microbial suspension temperature at which a microbial suspension is killed in a specific time (usually in 10 is killed in a specific time (usually in 10 minutes) minutes)

TDP depends not only on the nature of microbial TDP depends not only on the nature of microbial species but also on its stage, number and on species but also on its stage, number and on the local environment the local environment

Thermal death time Thermal death time (TDT) = the shortest time (TDT) = the shortest time needed to kill all microbes in a suspension For needed to kill all microbes in a suspension For most bacteria it averages 10-15 minutes at 60-most bacteria it averages 10-15 minutes at 60-65 °C65 °C

Osmotic pressureOsmotic pressure

Hypotony – the damage is prevented by the cell wall Hypotony – the damage is prevented by the cell wall HypertonyHypertony – mostly hinders microbes (therefore fruit is – mostly hinders microbes (therefore fruit is

candied, meat salted)candied, meat salted)

Higher osmotic pressure Higher osmotic pressure is endured by:is endured by:halophileshalophiles – halotolerant: enterococci (6,5% NaCl)– halotolerant: enterococci (6,5% NaCl) staphyloccoci (10% NaCl)staphyloccoci (10% NaCl)

– – obligate: halophilic vibria (in sea water)obligate: halophilic vibria (in sea water)mouldsmoulds – tolerate higher content of saccharose – tolerate higher content of saccharose

(in jams) (in jams)

Aspergillus nigerAspergillus niger

www.medmicro.infowww.medmicro.info

pH pH NeutrophilesNeutrophiles: growth optimum at pH 6 až 8 : growth optimum at pH 6 až 8 AlkalophilesAlkalophiles: e.g. : e.g. Vibrio cholerae Vibrio cholerae (pH 7,4-9,6) alkalotolerant: (pH 7,4-9,6) alkalotolerant: Proteus Proteus (it (it

splits urea), splits urea), Enterococcus Enterococcus (broad range of pH 4,8-11)(broad range of pH 4,8-11)On the contrary, there are microbes sensitive to extremes of pH: e.g. On the contrary, there are microbes sensitive to extremes of pH: e.g.

gonococcigonococci

AcidophilesAcidophiles: facultative: yeasts, moulds, lactobacilli (: facultative: yeasts, moulds, lactobacilli (>3)>3), coxiellae , coxiellae (tolerate low pH of phagosome)(tolerate low pH of phagosome),, obligate: obligate: Thiobacillus thiooxidans Thiobacillus thiooxidans (pH (pH <1)<1)

Microbes sensitive to low pH: mainly vibrios, streptococci, putrefactive Microbes sensitive to low pH: mainly vibrios, streptococci, putrefactive bacteria; low pH hinders most bacteriabacteria; low pH hinders most bacteria

Why sparkling water lasts longer? Because it has lower pHWhy sparkling water lasts longer? Because it has lower pHLow pH keeps spores from germinating Low pH keeps spores from germinating →→ botulism can be obtained botulism can be obtained

from from oil-oil-preserved mushroompreserved mushroomss or or preserved preserved strawberries, not from strawberries, not from pickled gherkinpickled gherkinss or mixed pickles or mixed pickles

Redox potential (rH) Redox potential (rH) - tendency of a chemical species to acquire - tendency of a chemical species to acquire electrons and thereby be and thereby be

reduced- l- level of rH depends both on the compositionevel of rH depends both on the composition of the environment of the environment

and the atmosphereand the atmosphere

AerobesAerobes – need high rH levels ( – need high rH levels (>200 mV)>200 mV)

AnaerobesAnaerobes – need low rH levels (≤0 mV) – need low rH levels (≤0 mV)Anaerobes are killed by OAnaerobes are killed by O22, aerobes without O, aerobes without O22 will live will live

Even so, anaerobes prosper both in nature and in our bodies – Even so, anaerobes prosper both in nature and in our bodies – thanks to the cooperation with aerobes and facultative anaerobesthanks to the cooperation with aerobes and facultative anaerobes

Anaerobes in the Anaerobes in the bodybody:: - - large intestine (99 % of bowel microorganisms) large intestine (99 % of bowel microorganisms)

- - vagina vagina - - oral cavityoral cavity (sulci gingivales) (sulci gingivales)

Radiation Radiation UV radiation UV radiation (maximum effect around 260 nm)(maximum effect around 260 nm)In nature airborne bacteria protects themselves by In nature airborne bacteria protects themselves by

pigmentspigments → they have color colonies→ they have color colonies Artificially: UV radiation for disinfection Artificially: UV radiation for disinfection

of surfaces, water, air; in PCR laboratories of surfaces, water, air; in PCR laboratories for destroying residues of DNA for destroying residues of DNA

Ionizing radiation Ionizing radiation ((XX and gamma radiation) and gamma radiation)For sterilizing disposable syringes, infusion sets, For sterilizing disposable syringes, infusion sets,

materials for dressing and sewing, tissue grafts, materials for dressing and sewing, tissue grafts, some drugs, even waste and food (not in EU)some drugs, even waste and food (not in EU)

Record holders for radiation resistance: Record holders for radiation resistance: Deinococcus radioduransDeinococcus radiodurans and and bacterial sporesbacterial spores

Toxic substancesToxic substances Their influence depends on the Their influence depends on the concentrationconcentration and and

durationduration of exposureof exposure

Various microbes markedly differ inVarious microbes markedly differ in relative relative resistance resistance to different types of toxic substancesto different types of toxic substances

In general (and contrary to drying):In general (and contrary to drying): G– bacteria are G– bacteria are more resistant to toxic substances then G+ more resistant to toxic substances then G+ bacteria bacteria (because of different structure of (because of different structure of bacterial cell wall bacterial cell wall → presence of enzymes in → presence of enzymes in periplasmatic space of G– bacteriaperiplasmatic space of G– bacteria))

For application is vital to know the effects of the For application is vital to know the effects of the particular substances used for particular substances used for disinfectiondisinfection

Bacterial cell wallBacterial cell wallG+G+ G–G– lipoteichoic acidlipoteichoic acid O-antigenO-antigen lipopoly-lipopoly- inner polysaccharideinner polysaccharide saccharidesaccharide lipidlipid AA (endotoxin)(endotoxin) mureinmurein

porinporin

outerouter membranemembrane

lipoproteinlipoprotein ENZYMES ENZYMES periplasmaticperiplasmatic space space

innerinner membranemembrane (G–)(G–)cytoplasmatic membrane (G+)cytoplasmatic membrane (G+)

Sterilization versus disinfectionSterilization versus disinfection

Sterilization = removal of all microorganisms Sterilization = removal of all microorganisms from objects or environmentfrom objects or environment

Disinfection = removal of infectious agents Disinfection = removal of infectious agents from objects and environment, respectively from objects and environment, respectively from the body surface from the body surface

Disinfection aims at breaking the chain of Disinfection aims at breaking the chain of infection transmissioninfection transmission

BiocidesBiocides = a new general term including also = a new general term including also disinfectantsdisinfectants

Types of disinfectantsTypes of disinfectants

1.1. Oxidizing agentsOxidizing agents

2.2. Halogens Halogens

3.3. Alkylating agents (aldehydes)Alkylating agents (aldehydes)

4.4. Cyclic compounds (phenolics)Cyclic compounds (phenolics)

5.5. Acids and alkaliAcids and alkali

6.6. Heavy metal compoundsHeavy metal compounds

7.7. Alcohols Alcohols

8.8. Surface active substances (QAS)Surface active substances (QAS)

9.9. OthersOthers

Relative resistance of different Relative resistance of different agents to biocidesagents to biocides

Enveloped viruses Enveloped viruses herpesvirusesherpesviruses

Some protozoa Some protozoa very susceptiblevery susceptible Trichomonas Trichomonas

Gram-positive bacteria Gram-positive bacteria StreptococcusStreptococcus

Gram-negative bacteria Gram-negative bacteria SalmonellaSalmonella

Yeasts Yeasts susceptiblesusceptible CandidaCandida

Moulds Moulds TrichophytonTrichophyton

Naked viruses Naked viruses enterovirusesenteroviruses

Protozoal cysts Protozoal cysts relatively resistant relatively resistant Giardia Giardia

Acidoresistant rods Acidoresistant rods MycobacteriumMycobacterium

Helminth eggs Helminth eggs AscarisAscaris

Bacterial spores Bacterial spores very resistant very resistant ClostridiumClostridium

Coccidia Coccidia CryptosporidiumCryptosporidium

Prions Prions extremely resistant extremely resistant agent of CJDagent of CJD

Universally effective biocides Universally effective biocides

Small, naked viruses: Small, naked viruses: oxidizing agentsoxidizing agents halogenshalogens aldehydesaldehydes strong acids and alkalistrong acids and alkali

Mycobacteria:Mycobacteria: oxidizing agentsoxidizing agentsaldehydesaldehydeslysollysolstrong acids and alkali strong acids and alkali

Bacterial spores:Bacterial spores: (oxidizing agents) (oxidizing agents) aldehydesaldehydes

strong acids and strong acids and alkalialkali

Recommended reading materialRecommended reading material Paul de Kruif: Microbe HuntersPaul de Kruif: Microbe Hunters

Paul de Kruif: Men against DeathPaul de Kruif: Men against Death

Axel Munthe: The Story of San MicheleAxel Munthe: The Story of San Michele

Sinclair Lewis: Sinclair Lewis: ArrowsmithArrowsmith

André Maurois: La vie de Sir Alexander FlemingAndré Maurois: La vie de Sir Alexander Fleming

Hans Zinsser: Rats, Lice, and HistoryHans Zinsser: Rats, Lice, and History

Michael Crichton: Andromeda StrainMichael Crichton: Andromeda Strain

Thank you for your attentionThank you for your attention