Classification & pathogenicity of Microbes

The microbial causes of human disease include viruses, Chlamydia, Richettsiae, Mycoplasmas, bacteria, fungi & protozoa. Basic features of these are included in table 1


Table 1. classification Of Microbes

Type of microbes Nuclic acid Multiplication Approx. Size (um) Seen by LM Cell wall Cytmenb. Senaitive antibiotic Other features

Intracellular Extracellur

Viruses DNA or RNA + - 0.01–0.3 No No No No Host cell may show inclusion

Chlamydia esc. Trachomates C. psittaci DNA + RNA +Multiplication by binary fission - 0.3 No No Yes Yes eg tetracycline Host cell may show chonilusin

Richettsiae eg coxiella, R.prowazeki DNA + RNA + by binary fission -occasional exception 0.3 Sometimes just visible by sp. Stains Rudimentary cell wall Yes Yes eg tetracycline Typheus transmission by arthropodes.

Mycoplasma eg. M.pneumonia, M. hominis DNA + RNA + Multiple. efementary badies + 0.12–0.3 ometines just visible by sp. Stains No Yes Yes eg tetracycline Remarry cells

Bacteria DNA + RNA ± + Multiply binoryfissi 0.5 – 0.8 long Yes Yes Yes Yes Rigid cell wall

Fungi DNA + RNA + + Larger than bact. (75 vong, >0.5 wide) Yes Yes (Thicker than bact. Cell + contains sterop) Yes No Sensitive to anti fungal Menles of plant lutno chlorgy

Protozoa DNA + RNA ± depends on particular species ± Laryer than fungi Yes Yes Yes Not usually -



Viruses differ greatly from all the other microbes as they consist essentially of only nucleic acid surrounded by a protein coat (capsid) & contain only one instead of two types of nucleic acid.

Chlamydiae & Richettsiae are also obligate intracellular parasites, have both DNA & RNA & multiply by binary fission. Mycoplasmas, bacteria & fungi can be cultured in cell free media unlike the above intra cellular microbes .

Protozoa pathogenic to man are divided into 3 main groups :

1. Sarcodina (amoebae) e g Entamoeba histolytica .

2. Sporozoa eg. Plasmodium falciparum, Toxoplasma gondii.

3. Mostigophora (flagellates) eg Trichomonas vaginalis, Giardia lambilia, Leishmania & Trypansoma sp.

Classification of bacteria :

There are 3 main group of bacteria :

1- Bacteria that are readily Gram – Stained

2- Acid – fast bacilli

3- Spirochetes'

Bacteria that are readily Gram – Stained:

These are classified into Gram positive (blue-purple) or Gram negative (pink-red) cocci or bacilli (Table 2)

After the application of the methyl violet dye, Gram positive bacteria stain blue & this color is retained in spite of decolorization with acetone (or alcohol)and Gram negative bacteria initially stain blue after the methyl violet is applied but the color is lost after the application of acetone (or alcohol). They then take up the pink counter stain (Carbol fuchsine, methyl red or saffronin).

The reason for the difference in color after gram staining is not fully understood, but it is probably related to the large amount of mucopeptide & teichoic acid in the cell walls of Gram positive bacteria. The fact that Gram positive bacteria are more acidic than Gram negative bacteria may account for their greater affinity for a basic dye. Even more important may be the greater permeability of Gram negative cell walls which allow the methyl violet – iodine dye complex to diffuse out after treatment with acetone more readily than cell walls of Gram positive bacteria.

Within each sub group, there are aerobic or anaerobic examples . The majority of bacterial pathogens can grow either aerobically or anaerobically i.e. facultative anaerobes such as Staphylococcus aureus or Escherichia coli in table 2 these have been included as aerobes. There are a few bacterial species which are strict aerobes such as Pseudomonas aeruginosa which will not grow at all anaerobically. Some bacterial species are strict anaerobes such as Clasteriduim tetani or Bacteroides fragilis, which will not grow at all aerobically .

Exceptional Gram negative stainable bacteria include Legionella pneumophillia & Borrelia vincent. Legionella pneumophillia requires prolonged staining with the counter stain to be seen in tissues, although it appears readily as Gram negative bacilli in smears made from colonies on agar media. Borrelia vincent is the only spirochete pathogen that is easily seen by a Gram stain.

Acid- fast bacilli :

Mycobacteria species are not readily seen by a Gram stain. Zheil-Nelseen ( ZN) or other acid- fast stains are required for staining these organisms which have cell walls containing abundant lipids. Examples include Mycobacterium tuberculosis & Mycobacterium leprae .

Spirochetes :

They are thin walled spiraled flexible organism which are motile by means of an axial filaments and they are not seen in a Gram stain (except Borrelia vincent) but may be seen either by dark ground illumination microscopy or in a silver stain under the light microscope. Borrelia spirochaetes in the blood may also be seen in a Giemsa stain.

The three groups of spirochetes include :

1. Treponema : Spirochetes with regular spirals, about 1um apart from each other, 5 – 15um long & about 0.2um wide e.g. Treponema pallidum .

2. Leptospira : have tightly spirals, 5 – 15um long & about 0.1um wide. There is often a hooked end eg Leptospira ictero haemorrhagiae (Weill's)

3. Borrelia : Large spirochaetes, 10 – 30um long & about 0.3um wide, with irregular spirals 2 – 4um apart from each other eg Borrelia recurrentis (a cause of relapsing fever.

Table 2 : Simple classification of Gram stainable bact. pathogens :-


Bacteria Genus Sp. examples

G positive Bacteria.

• Cocci

o Aerobic





o Anaerobic



• Bacilli

o Aerobic





o Anaerobic



Clusters …. Staphylococcus



Chains/ pairs …. Streptococcus pneumoniae

….. Strept





….. Sporing

….. Non Sporing







….. Sporing



….. Non Sporing



…. Staphylococcus aureus



Staphylococcus allbus (S.epidermidis)

…. Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus uridans, Enterococcus faecalis

….. Streptococcus putridus





…. Bacillus - B.anthracis

…..Corynebacterium – C. diphtherum – Listeria – L.monacytogenes – Nocardia – N.asteroides



…..Clostriduim- C.tetain c.cvelchie (perfrorigens)

….Propionbacterium - P. acnos - Actinomycen - A.israelic



G negative Bact.

• Cocci

o Aerobic

o Anaerobic



• Bacilli

o Aerobic

o Anaerobic



… Paris

…





…a. Enterobacteria











b. Pseudomonas

c. Vibrios



d. Parvobact







e. Legionella

f. Spirillum

Amaerabic –

Bacteroides –



… Neisseria – N. meningitis – N. gonorrhoea

…Veillonella





e.g. Escherichia – E Coli

Klebsiella – K.aerogenes

Proteus – P.mirabilis

Serratia – S.marcescens

Salmonella – S.typhi

Shigilla – Sh. sonnei

Pseud – P.aeruginosa

Viberio – V. cholera

Compylobacter – C. Jejuni

Haemophilus – H.influenzae

Brucella – B. abortus

Bordetella – B.pertussis

Pasterella, yersini –

L.pneumophilia –

S.minus



B.fragilis

Classification of viruses:

The classification of viruses depends on several factors including the type of nucleic acid present, the arrangement of the capsids into a circular(icosahedral), helical or complex symmetry, the number of capsomeres, the shape of the virus particle & whether the vision is naked or enveloped .

DNA viruses are:

Pox & papova, Herpes & adeno virus & the remaining viruses & the remaining viruses are RNA viruses .

Classification of fungi :

There are 4 main groups of pathogenic fungi moulels (filamentous fungi) true yeasts yeast – like fungi & dimorphic fungi

1- Filamentous fungi :

Theses grow as long filaments called hyphae & the branched hyphae intervene to form a mycelium. Reproduction is by spores including sexual spores which are used for identification. Culture on Sabaroud's powdery colonies due to the presence of spores eg Trichophyton mentagrophyets .

2- True yeasts :

These are unicellular round or oval fungi . Reproduction is by ludding from the parent cell . Cultures creamy colonies e.g. Cryptococcus neoformans .

3- Yeast like fungi:

These are like yeasts since they may appear as round or oval cells & grow by budding . They may also form long non branching filaments known as pseudohyphae e.g. Candida albicans.

4- Dimorphic fungi :

These grow as yeast forms in the body & at 37oC on culture media. They also form mycelia in the environment & on culture media at 22oC eg Histoplasma cupsulatum.

Fungi can also be classified according to whether they cause superficial or deep mycoses in infected patients .

Pathogenesis : Factors affecting the virulence & Spread of Microbes :

The pathogenicity of a microbe depends on host as well as on microbial factors . Host factors include the age of the patient, genetic factors, general host defenses & local host defenses .

Lock's postulates :

1. The particular microbe is always associated with a given disease .

2. The microbe may be isolated in the laboratory from specimens from a patient with the disease .

It is possible to produce a similar disease in animals by inoculation of the microbe into animals. Mycobacterium tuberculosis may be an example where these 3 postulates may be fulfilled.

Factors affecting "Virulence":

The main known factors that affect virulence are concerned with pathogenicity such as toxins & capsules in bacteria . It has also become apparent that the virulence of bacterial stains may also depend on the presence of transmissible genes contained in plasmids or mediated by bacteriophage.

The toxins produced by Corny diphtheriae & the erythrogenic toxin produced by Streptococcus pyogenes strains in scarlet fever patients are dependent on genes mediated by temperate phages. The fact that particular microbes appear to be more or less virulent at different times, might be due in part to the presence or absence of these types of transmissible genes.

Factors affecting spread:

Epidemiological factors affecting the host are relevant to the spread of microbes including the numbers of susceptible individuals in a geographically defined area, the proximity of the individual to each other & to the source of infection & the presence of other factors necessary for the transmission of infection such as the correct climate or season, the presence of an essential arthropod vector, etc.

Microbial factors that affect the spread depend partly on the virulence of the microbe & partly on the ability of the microbe to survive or multiply in a given inanimate environment or on the hands of patients or hospital staff or in animals arthropods. Carrier states clearly aid the transmission of bacteria. Gram positive bacteria survive well in dry environments while Gram negative & some spirochetes survive lest in moist situations .

Microbes are either transmitted horizontally i.e. between individuals of the same generation (such as the plague bacillus) or vertically i.e. between individuals of different generation (such as cong. Rubella for mother to infant).

Infection either endogenous from the patient's own flora or exogenous from a source outside such as another patient or person, an animal, a vehicle or fomite .

Mode of transmission of microbes include:

1. Direct contact such as N. gonorrhoea .

2. Ingestion such as V. cholerae.

3. Inoculation such as injury transmitting hepatitis B, mosquito bite transmitting malaria .

4. Inhalation such as with measles, Rhino viruses or Mycobacterium tuberculosis.

The Use of the Clinical Microbiology Laboratory (General Principles )

* Collection of Clinically Relevant Specimens:


The best use of the laboratory involves sending only relevant specimens so that work can be done on a reasonable no. of specimens especially when economic situation of the country is greatly limited

Examples of unnecessary investigations include routine microscopy and culture of urine specimens from all non catheterized adult patients in hospital without symptoms suggestive of urinary tract infection.

The role of the consultant clinical microbiologist:

A- Regular contact with clinical colleagues to ensure the appropriate investigation for the clinical conditions, and that good quality specimeas are sent

Organize the laboratory so that the relevant investigations are carried out reliably, safely and economically. Important results are communicated to colleagues and discussed.

B- As regard consultation on the investigation and management of patients with infection problems. Seeing patient on the words, temp. charts, drug shets. Etc., together with clinical colleagues

Discussing difficult clinical problems and management of out breaks of infectious disease

C-As regards Control of hospital infection: design implementation of policies on the use of antibiotics, isolation procedures, sterilization and disinfection

D- As regard teaching research: Education of medical staff about infections, the use of antibiotics, disinfectants. Etc.

-Research on epidemiology, diagnosis, treatment or prevention of infections.

Provision of essential clinical information:

The information routinely required in laboratory request: age, brief details of the main clinical condition , date of onset of the illness, antibiotic therapy, history of recent travel abroad, suspected source of infection.

Prior Discussion with microbiologist.

To achieve the best use of the laboratory certain types of investigation need to be discussed with the microbiologist. These include, assay of antibiotics the isolation of viruses, molecular biology tests, and the investigations of possible cross infection incidents

Proper Sampling in Clinical Microbiology Laboratory

Collection of good quality specimens:

Depends on

1- The optimal time of specimen collection.

2- The correct type of specimen

3- Well collected specimens with minimum contamination from normal flora of the patient or the person collecting the specimen.

4- Adequate amounts of each specimens and appropriate no. of specimens

5- Clearly labeled safe specimens

1- Optimal time of collection of collection of specimens:

-specimens for the culture of bacteria collected before the start of antibiotic therapy

-Blood cultures and blood films for malarial parasites are best collected just as the patient’s temp starts to rise, however, when infective endocarditis is suspected, three blood culture sets collected with 24 hour irrespective of patient temp.

- Specimens for virus isolation are most likely to give positive results when collected during the most acute stages of the disease

-Serology is satisfactory when four fold or greater rising antibody titer is demonstrated in pained sera.

The 1st serum sample as early as possible in the disease course. Second in the convalescent stage

2-Correct types of specimens:

Examples:

Bacterial meningitis-------blood cultures CSF culture

Suspected gonorrhea -------cervical, urethral and rectal swab should be collected rather than high regional swabs.

3-Well collected specimens with minimum contamination from the normal flora:

Poor quality specimens include saliva instead of sputum or a salivary – mucoid sputum sample instead of a muco purulent sputum.

-Mid stream urine need careful collection to a void excess contamination by genital flora.

-A throat swab should not touch the buccal mucosa and the tongue depressed by a spatula.

-Vaginal speculum should not be wet with antiseptic solution during collection of high vaginal swab with care not to touch the lower vagina or perineum.

-Strict septic and antiseptic techniques are used for blood and CSF cultures to avoid contamination from skin flora or from the doctor.

4-Adeqate amounts of appropriate number of specimens:

The volume of blood for culture from adult

-5-10 ml per bottle and in children and neonates 1-5ml per bottle.

-Collection of early morning sputum specimens, and collection of adequate amount of early morning urine specimen for 3 successive days is required for the isolation of M.T.B.

-Patients with diarrhea ---at least 2 specimens of faeces is collected for culture of Salmonellae or Shigella.

-Serological investigations usually require paired sera.

5-Clearly labeled and safe specimens:

Specimens for microbiological investigations should be placed in leak – proof containers, and each container should be enclosed in plastic bag.

The hazards to staff handling leaking container s include acquiring enteric infection from feces, T.B. from sputum of an open case of pulm. T.B. and viruses s such as HCV, HBV, HIV, from leaking blood.

Transport of specimens to the laboratory

Many pathogenic organisms don’t survive for long in clinical specimens kept at room temp. Examples include Gonococci, Haemophilus, Bacteroides, anaerobic cocci and most viruses.

On the other hand, some organisms contaminating specimens from the normal flora such as Coliform and Coagulase negative Staphylococci, may rapidly grow in specimen kept at room temp.

-Urine or sputum specimens should reach the laboratory within 2hours of collection when even possible. If delay are expected immediately inoculated into transport media.

-Transport media used:

Stuart’s transport media ----- for pus or swabs for bacterial culture when delays in transport.>1/2hour or when Neisseria infections are suspected. However the inoculated transport media should be sent to the laboratory within 4h.

The investigation of eye, genital tract is best carried at the bed side when suitable culture media are directly inoculated.

-Cerebrospinal fluid(CSF) not refrigerated since other wise Meningococci may rapidly die.

-Viral transport media is necessary for virus isolation, and also for Chlamydia isolation.

Specimens for virus isolation are kept at –70ºC till time of transferring the appropriate cell line which support growth of the possible virus or Chlamydia.

Basic Laboratory procedures for microbiological diagnosis

Laboratory procedures for microbiological diagnosis Include the following steps:

-Naked eye examination of Specimens

-Microscopy.

-Detection of microbial antigens.

-Isolation of microbes

- Antibiotic sensitivity

-Serology.

-Molecular biology techniques.

-Gas – liquid chromatographic techniques.

-Skin tests.

I. Naked eye examination of specimens:-

This helps to determine whether a specimen is suitable or no.

- A saliva sample instead of an expectorated sputum sample should be discarded.

-Turbid CSF, is an immediate evidence of infection

-A foul smelling pus specimen may suggest presence of anaerabes.

-A rice water stool sample may indicate Vibrio cholera infection

-Anchory sauce sputum sample would suggest invasive Amoebiasis in lungs.

-Sulphar granules in pus would indicate Actinomycosis.

II. Microscopy:

1-Wet preparation for light microscopy in examination of CSF, urine, body fluid for evidence of pus cells, and organisms.

-Vaginal secretion-- Trichomonas and Candida .

-Skin, nail , hair (in KaOH) -evidence of fungus

-Dark ground illumination to look for Spirochates, Treponema pallidum in suspected 1ry or 2ry .

2-Gram stained smear: It may help of saving life.

-Important in rapid diagnosis of bacterial meningitis on exam of CSF deposit.

-Diagnosis of Strepto pneumonia in sputum smear

-Performed in serious septicemia when a blood culture bottle is flagged positive.

- Identification of colonies appearing on culture media

-Gram – stained smear may give positive results al though the subsequent cultures are negative as a result of given antibiotics

-In Vincent's angina: stained smear the only means of diagnosis

3-Acid fast stain of sputum allow rapid diagnosis of open pulmonary tuberculosis demonstrating acid fast bacilli. In other, clinical specimens such as urine, peritoneal fluid, CSF it lacks sensitivity.

4-Immunoflourescent microscopy is important for rapid diagnosis of viral infection e.g. Respiratory syncytial virus (RSV) in infants and children, Herpes simplex, cytomegalovirus (CMV) in urine throat swab, Rabies in brain biopsy specimen, Clamydia trachomatis in conjunctival scrapings. Also, it can be used in serological antibody test e.g Fluorescent Treponema antibody and fluorescent amoebic antibody.

5-Eectron Microscopy:

Mainly used for rapid diagnosis of rota virus or herpes infection, CMV in neonatal urine specimens as the virus voids in urine in large amount.

III- Detection of microbial antigens:

Becomes increasingly important in recent years.

- Immunoelectrophoresis . e.g Pneumococcal polysaccharide antigen may be detected in sputum, serum, urine of patients by immunoelectrophoresis, when patients have already given antibiotics where as conventional sputum blood cultures are negative.

- Hepatitis B surface antigen (HbsAg) is commonly detected using ELISA or latex test

-Cryptococcal antigen in CSF in patients with cryptococcal meningitis with latex.

-Rota virus antigen using enzyme linked immunosorbant assay(ELISA) in feces of diarrheic children infected with rota virus.

-Chlamydia trachomatis antigen using ELISA in conjunctival scrappings in patients with active trachoma.

-Detection of Meningococcae, Hemophilus, Pneumococcae antigen in CSF specimens by latex

-Commercially available monoclonal specific antibodies for detection of antigen in clinical specimens or cultures including various Streptococcal, Staphylococcal species, Neisseria, Candida spp. Chlamydia trachomatis, and Rotariruses, CMV, Herpes, Adeno viruse, RSV, Influenza viruses.

IV- Isolation of microbes

Is the most reliable way in which a diagnosis can be confirmed and for obtaining antimicrobial susceptibility results.

-Isolation of bacteria or fungus from specimens such as Blood, CSF (which are normally sterile) are easy to interpret. While, bacterial or fungal isolation from specimens collected from sites with normal flora are often difficult to interpret.

-Choice of media is important according to type of specimen and suspected organism.

-Virological or chlamydial isolation methods. Need preparation of the cell line required for support growth of suspected infecting virus.

For Chlamydia -----Vero, Maccoy line

For Adeno - Vero , HEP

For CMV- Human diploid fibroblast.

For Influenza------ chick embryo.

V-Antimicrobial susceptibility testing

1-Disc diffusion tests

Limitation of disc diffusion tests:-

-Not applied to slowly growing, Fastidious organisms or anaerobes .

-Mycobacterial and fungus susceptibility testing requires specific techniques

-The reported sensitivity tests results not applied to clinical sites infections, e.g Salmonella typhi to aminoglycosids.

-Not related to the achieved serum levels or body fluid levels of antibiotics.

-Bacteriostatic measures only.

-Can’t be applied to certain antibiotics such as polymyxines.

2-Dilution susceptibility testes:-

Micro minimal inhibitory and minimal bactericidal activity methods.

Methods

-Broth dilution tests

-Agar dilution method.

Application:-

-Serious infection where endpoint concentration is ended

-Disc diffusion yield inter mediate susceptibility

-Life threatening infection due to organisms with unpredictable susceptibility pattern.

-Fastidious or slowly growing organisms.

-Failure of antibiotic therapy

-Serious infections caused by organisms susceptible only to toxic agents

Limitations

-Difficult

It needs the knowledge about the achievable level in serum or body filmed

3- Automated method

4-Antimicrobial concentration gradient methods

-A serial antibiotic dilations are incorporated into the agar.

-E test

VI-Serology

Ten days or longer has to pass before arising antibody demonotrated

in chronic infections. e.g , Brucellosis serology is often available

-IgM antibody may indicates recent infection (e.g Rubella)

-ELISA technique widely used nowadays for detection of ab

Immune status of the patients immunization should be taken in considerations.

VII-Molecular biology techniques:

Increasingly important for rapid diagnosis of infections for epidemiological investigations and for monitoring antimicrobial therapy

Also, important for research on the pathogenesis of infection, the developmental new vaccines and immune therapeutic agents.

VIII Gas Liquid chromatographic techniques:

Become increasingly useful for the rapid detection of anaerobic infections

Specimens of pus from abdominal, gynecological or brain abscesses may be shawn to have multiple volatile fatty acids present which indicate anaerobic infection with a few hours of collection of the specimen of pus and this may affect decisions about the chemotherapy of infection.

IX- Skin test:

Of limited value for diagnosis of infection ex:

-Mantaux skin test for tuberculosis.

-Histoplasmin test for Histoplasma infection.

-Casoni test

-Schick test