Normal Flora

  Dermatology BM 2023 Lecture 5   Dr Tim Scott-Taylor Health and Human Sciences

Topics

Information covered;   relations to host significance of flora     position and density (various areas) bacteria involved benefits of bacterial flora potential problems

Learning Objectives

 Where normal flora comes from  Details of the prominent bacteria on skin  How they attach and where they reside  benefits of normal flora  Significant examples of opportunistic infections

Normal Flora

Present on all exposed skin surfaces Only tissues and certain internal surfaces are sterile Almost exclusively bacteria (protozoa) Usually benign, but potential for opportunistic infection

Surfaces

   Internal tissue: eg blood, brain, muscle normally free of microrganisms Surface tissues: eg skin and mucus membranes constantly in contact environment readily colonised by some species Internal mucosa: eg urethra, vagina, respiratory tract low density specific organisms The mixture of organisms regularly found at surface anatomical sites is referred to as the normal flora or microbiota

Microbes

     Microbes; microscopic; found in virtually all environments viruses bacteria fungi parasites.

Man is surrounded by microbes. Most of these are innocuous and are unlikely to pose a major threat to animals. saprophytes are harmless microbes that live on inanimate material and derive their nutrition from these environmental sources.

symbionts require a human or animal host to survive and multiply. Symbiosis does not distinguish between relationships that are harmful or beneficial

Symbiotic Relationships

    Mutualism: two organisms from different species living in close proximity to one another and relying on one another for nutrients, protection or other life functions. Both organisms involved benefit from the relationship.

Commensalism: two organisms from different species living in close proximity to one another, in which one member is unaffected by the relationship and the other benefits from it Parasitism: two organisms from different species living in close proximity to one another, in which one member depends on another for its nutrients, protection or other life functions. The parasite benefits from the relationship while the host is harmed by it Amensalism: two organisms from different species living in close proximity to one another, where one of the members suffers as a result of the relationship while the other is unaffected by it

Commensalism

     normal flora are commensals that derive food and shelter from the host. They normally do no harm to the host. The host may benefit from the presence of the microbe. relationship also described as mutualism. No harm is done to the host. Commensal microbes colonise the host Infection implies that harm is done to the host i.e. causes disease. A microbe that causes disease is a pathogen. Usually the host will manifest an inflammatory response to a pathogen, but not to a coloniser at a normally non-sterile body site

Pathogen

     a microbe that can initiate infection, often with only small numbers strict pathogens i.e. will always cause disease eg Bacillus anthracis (anthrax). Some pathogens may sometimes almost behave like commensals, eg

Salmonella typhi

(typhoid) (carrier state).

Whether a microbe behaves as a pathogen i.e. causes disease, depends on the properties of the microbe and the host. There can be a fine balance between microbial and host properties; Immunity Resistance microbiota Virulence Disease

Nature of Normal Flora

  A few Archaebacteria in the gut:

Methanobrevibacter smithii

Some prokaryotes,

Candida albicans

in mouth

Pneumocystis carinii Entamoeba gingivalis

in pharynx between teeth

Tooth Amoebas

    

Entamoeba gingivalis

microscopic parasites 10-35 μm Live in crevices between teeth and gums. Brushing does not remove them Beneficial; eat mouth bacteria only harmful when v. numerous lack of oral hygiene No cysts are formed; transmission is entirely by oral-oral contact.

Nature of Normal Flora

 A few Archeabacteria in the gut  Some prokaryotes, Candida albicans in mouth

Pneumocystis carinii

Entamoeba gingivalis in pharynx  But microbiota almost exclusively

bacterial

Numbers

    Human body = ~10 13 cells Bacteria; 10 10 10 12 in the mouth on the skin 10 14 in intestine Far in excess of number of eukaryote cells in all organs Skin low density 100-1000s/cm 2 ; except axillae high moisture level groin toe Density varies; age sex diet nutrition

Bacterial Staining

Bacteria distinguished primarily on shape; staining; cocci / bacilli gram +ve / -ve

Gram stain

Divides bacteria into

Gram positive – blue/purple Gram negative- red

Hans Christian Gram, Danish bacteriologist devised stain in 1882

Gram Staining

Gram negative Gram positive

Gram’s Stain Procedure

1. Place a slide with a bacterial smear on a staining rack. 2. Stain the slide with

crystal violet

for 1-2 min. 3. Pour off the stain. 4. Flood slide with Gram's iodine for 1-2 min. 5. Pour off the iodine. 6. Decolourize the slide briefly with acetone (2-3 seconds). 7. Wash slide thoroughly with water to remove the acetone 8. Flood slide with safranin counterstain for 2 min. 9. Wash with water. 10. Blot excess water and dry in hand over bunsen flame.

How Does it Work?

   Gram didn't know - he simply worked empirically Gram reaction is based on the structure of the bacterial cell wall In Gram-positive bacteria, the

purple crystal violet stain

layer of peptidoglycan which forms the outer layer of the cell is trapped by the • In Gram-negative bacteria, the outer membrane prevents the stain from reaching the peptidoglycan layer in the periplasm. The outer membrane is then permeabilized by acetone treatment, and the

pink safranin counterstain

is trapped by the peptidoglycan layer.

Bacterial Cell Walls

Lipoteichoic acids traversing wall and anchored in basement membrane Bacterial cell walls porin polysaccharide (O antigen) lLipid A cell wall lipoprotein peptidoglycan Lipid bilayer plasma membrane with integral proteins Gram-positive Lipid bilayer plasma membrane with integral proteins Gram-negative

Four Basic Types

Gram + cocci Gram - cocci e.g. Staphylococcus aureus Gram + rod e.g. Neisseria Gram - rod e.g. Bacillus cereus e.g. Escherichia coli

Bacterial Shape

Common Bacteria in Flora

BACTERIUM

Staphylococcus epidermidis Staphylococcus aureus*

Corynebacteria Mycobacteria

Streptococcus pyogenes* Streptococcus pneumoniae* Neisseria sp. Haemophilus influenzae* Escherichia coli * Streptococcus mitis Streptococcus salivarius Streptococcus mutans* Enterococcus faecalis* Bifidobacterium bifidum Lactobacillus sp.

++ = nearly 100 percent

Skin ++ + ++ + +/ Eye + +/ + +/ +/ + +/ +/-

+ = common

Nose ++ + ++ +/ +/ + + +/ ++ + + ++

+/- = rare

+ + + + + ++ ++ ++ + Intestine + ++ + + +/ ++ +/ ++ ++ Urethra ++ +/ + + + + + + Vagina ++ + + +/ +/ + + + + ++

* = potential pathogen

++

Specific Organisms

Skin surfaces Oral cavity Large bowel Nose Nasopharynx & upper resp. tract Vagina staphylococci, corynebacteria and streptococci streptococci, staphylococci, corynebacteria, anaerobic organisms, yeast (fungi) anaerobic bacteria e.g.

Bacteroides

sp., a number of coliforms e.g.

Escherichia coli

, and faecal streptococci e.g

. Enterococcus

sp. staphylococci, streptococci, corynebacteria streptococci, staphylococci, corynebacteria,

Neisseria

sp.

Lactobacillus

sp,

Bacteroides

sp,

Enterococcus

sp.

Skin

     Not a great habitat; dries out, constantly being shed, secretions include fatty acids (lower pH to 4-6) and salt. Some skin regions better habitats than others: scalp, ears, underarms, anal region are all especially good Bacteria that can grow on skin must be able to survive these conditions. Typical bacteria: normally a pathogen: but infection via surgical implants and catheters

Staphylococcus epidermidis

, not

Propionobacterium acnes

; lives in sweat glands, hair follicles, not eliminated by washing skin.

Staphylococci

Gram positive cocci in clusters • Greek • •

staphyle S. aureus

: coagulase test clot plasma

S. epidermidis

= bunch of grapes cause of soft tissue infections toxic shock syndrome (TSS) scalded skin syndrome. : common on skin, coagulase-negative

Corynebacteria

Gram positive rod (bacillus) Pallisades Chinese writing • common on skin and in GI tract • irregular pleomorphic with metachromatic granules • grey / black colonies on tellurite medium •

C. diphtheriae

throat pathogen potent toxin

Streptococci

Gram positive cocci in chains • Streptococci occur on all skin and mucosal surfaces • glycocalyx: adherence to plastic and cells resistance to phagocytosis and antibiotics.

•

Streptococcus pyogenes

common on skin but causes erysipelas, scarlet fever, rheumatic fever

Conjunctiva

 Not completely sterile, numbers of bacteria is small Lachrymal secretions contain lysozyme Blinking continually wipes away bacteria 

Staphylococcus epidermidis

Corynebacteria Blepheritis Conjunctivitis Specific attachment to receptors sialic acid residues dominant Haemophilus Chlamydia

Respiratory Tract

Nares (nostrils) are heavily colonised > 200 species Staphylococcus epidermidis Corynebacteria Staphylococcus aureus dominant (20% of population) Upper respiratory tract most non-pathogenic but some pathogens Lower respiratory tract also highly colonised non-haemolytic Streptococci Neisseria Streptococcus pneumoniae Haemophilus influenzae Mucus action of cilia tissue damage disease

Urogenital Tract

   Upper urogenital tract; Anterior urogenital tract; Vagina; normally sterile action of urine

Staphylcoccus epidermidis Escherichia coli

Corynebacteria Staphylococci Streptococci Colonise soon after birth

Lactobacillus acidophilus

: at puberty lactic acid prevents establishment other bacteria

Mouth

   Favourable habitat; lysozyme nutrients and secretions epithelial debris saliva 10 4 -10 9 /ml organisms

Streptococcus salivarious

; 98% predominant until teeth erupt Flora increasingly complex

,

anerobes, Bacteroides

Streptococcus mutans Streptococcus sanguis

Dominant , caries

Streptococcus pyogenes

rheumatic fever damaged heart valves

Dental Caries

   Initiated by normal flora,

Streptococcus mutans

Dental plaque; consists 60-70% cells salivary polymers bacterial extracelular products Initiated by glucosyl transferase, surface enzyme initial attachment to tooth via salivary glycoprotein creation of glycan biofilm = bacterial capsule lactic acid from dietary sugars demineralises enamel Lactobacilli

Actinomyces israelli

secondary proteolytic invaders

Normal Gut Flora

relatively few bacteria in anterior bowel increasing diversity and density of bacteria in posterior bowel

Escherichia coli

Gram negative rods (bacilli) • type faecal bacterium (enterococus in US) • opportunistic pathogen in urogenital canal •

E. coli

0157: haemorrhagic colitis, uncooked meat

GI Tract

    digestive enzymes and stomach acid kill bacteria small intestine has few bacteria, colon huge population 1/3 of faeces is bacteria up to 1000 organisms/gram over 300 different species

E. coli

is only 0.1% of total population anaerobic Bacteroides most abundant; ~ 25% microbiota bacteria in colon divide every 12-24 hours on average, much slower than laboratory batch culture rates.

GI Tract

      high flow rates make small intestine difficult to colonize, concentration of bacteria remains low, ~10 6 /ml takes food ~3-5 hours to move through small intestine takes food 24-48 hours to travel through the colon slow flow rates allow bacterial multiplication, 10 12 -10 13/ ml 30-50% of contents, ~2-3 lbs weight = bacteria bacteria breakdown complex polysaccharide; xylan called colonic fermentation cellulose pectin colon an organ of digestion where normal flora does most of the work

Tissue Tropism

Normal flora exhibit a tissue predilection for colonisation Could be due to; - supply of a specific essential growth factor - construction of a biofilm some bacteria are able to colonise the biofilms of others, most biuofilms are a mixture of bacteria - a specific receptor present at some sites

Examples of Tissue Tropism

BACTERIUM

Staphylococcus epidermidis Staphylococcus aureus Streptococcus pyogenes Neisseria gonorrhoeae Streptococcus mutans Streptococcus salivarius Vibrio cholerae Escherichia coli

TISSUE

Skin Nasal membranes Throat Urogenital epithelium Tooth surfaces Tongue surfaces Small intestine epithelium Small intestine epithelium For some of bacteria in normal flora the attachment factors are precisely known

Attachment Factors

BACTERIUM

Streptococcus pyogenes Streptococcus mutans Streptococcus salivarius Staphylococcus aureus Neisseria gonorrhoeae Escherichia coli Vibrio cholerae

BACTERIAL LIGAND TISSUE RECEPTOR ATTACHMENT SITE Protein F fibronectin Glycosyl transferase Salivary glycoprotein Lipoteichoic acid Cell-bound protein Methyl- alanine pili Type-1 fimbriae Methyl-alanine pili Unknown fibronectin Pharyngeal epithelium Pellicle of tooth tongue epithelium Mucosal epithelium Glucosamine-galactose cervical epithelium mannose Fucose and mannose Intestinal epithelium Intestinal epithelium

Gnotobiotic Animals

   Germfree, axenic; to study effect of normal flora easy to produce germfree birds; sterilize shell, use sterile incubator Germfree mammals; air, food, water sterilized cesarean section isolation chambers  Germfree animals are less healthy than animals with normal flora;  Greater vitamin requirements for K and B complex     lower cardiac output much more susceptible to pathogens much smaller infectious dose required to initiate an infection Live shorter lives

Benefits of Normal Flora

     Synthesise and secrete vitamins; germ-free deficient Prevent colonisation by pathogens germ free 10 salmonella vs 10 6 lactobacilli maintain high pH vitamin K vitamin B12 oral cavity, skin gut vagina Antagonise other bacteria; inhibit / kill Stimulate certain tissues; poorly developed immune system Stimulate cross-reactive antibody immunise against pathogens fatty acids peroxide bacteriocins Peyer’s patches caecum

Disadvantages of Normal Flora

1. Body odour      body odour originates from the skin decoposition of secretions of apocrine sweat glands located primarily under arms and in the groin

Corynebacterium tenuis

and

C. xerosis

in particular best eliminated through good hygiene fungal infections such as athlete's foot also odourous

Antibiotic Resistance

      MRSA : Methicillin Resistant

Staphylococcus aureus

resistant to all commercially available antibiotics, including methicillin and vancomycin carried in the noses of health care workers and transmitted from patient to patient major cause of surgical wounds and systemic infections antibiotic resistance is transferred to other organisms recent NHS directive to alcohol wash hands between beds has cut incidence by 50%

Opportunistic Infection

Some commensals can act as pathogens when host’s defences are weakened or immunocompromised  infection caused by a normally benign commensal eg infection with

Candida albicans

(yeast) a normal commensal of mouth and gut in immunosuppressed leukaemia patients eg infection with

Pneumocystis carinii

a low virulence fungus during mmunosuppresion due to HIV

Opportunistic Infection

Some organisms that are commensals at one body site may be pathogens at another body site eg

Staphylococcus aureus

in the nose (commensal) in a post-operative wound infection (pathogen) eg

Escherichia coli

in GI tract (commensal) in urinary tract causes UTI (pathogen).

Dental Caries

    Teeth in skulls from Europeans prior to the 1500's showed remarkably well-preserved teeth. Once

sucrose

, a dissacharide from cane sugar, was introduced tooth decay became widespread

S. mutans

produces a thick capsule of dextran. The gooey polysaccharide forms a biofilm and allows other bacteria to attach

Dental Plaque accumulates

S. mutans

uses lactic acid fermentation exclusively as its catabolic pathway. Acids attack tooth enamel

Ulcers

     Stomach acid attacks duodenal lining; ulcers related to stress; 1980s Warren and Marshall discovered antibiotics reduced ulceration, awarded Nobel Prize Medicine 2005 30-50% of human population carry

Helicobacter pylori

Spiral shape and flagella helps bacterium burrow into protective mucous lining. Enzyme urease converts urea into ammonia and bicarbonate, neutralizes stomach acid ammonia protease catalase phospholipases toxic to duodenal epithelial cells

H. pylori

and Gastric cancer

   bacterium categorized as group I carcinogen by the International Agency for Research on Cancer (IARC) Gastric carcinoma MALT lymphoma (mucosa associated lymphoid tissue associated with

H. pylori

Two related mechanisms proposed; 1. free radicals production increases rate of cell mutation 2 . TNFα, interleukin 6, inflammation alter cell adhesion proteins and mutate tumor suppressor genes

Summary

 Bacteria perform physiological, nutritional and protective functions in the human body.  Maintaining a balance is crucial Flora consists of ecosystems consequences of disruption unpredictable. antibiotics tissue damage medical procedures changes in diet  normal flora is complexity and understanding of function limited eg < 1% of bacteria grow on laboratory media > 99% the microbial world unexplored