Common Microorganisms Cause Wound Infections

Wounds subject to infection can be surgical, traumatic, or physiologic.

Sources of infection include (1) the patient’s own normal flora; (2)
material from infected individuals or carriers that may reach the wound
on fomites, hands, or through the air; and (3) pathogens from the
environment that can contaminate the wound through soil, clothing, and
other foreign material. Examples of such infections include
contamination of a penetrating stab wound to the abdomen by
colonic flora, contamination of a clean surgical wound in the operating
room with S. aureus spread from the flora of a perineal carrier, and
introduction of spores of Clostridium tetani into the tissues on a splinter.
Possible pathogens
Gram positive
Examples :Staphylococcus aureus, Streptococcus pyogenes,
Enterococcus species [Vancomycin-resistant Enterococci(VRE)]
Clostridium perfringens.
Gram negative
Examples Pseudonomas aeruginosa, Proteus species, Escherichia coli

 Staphylococcus aureus is the commonest pathogen isolated from

subcutaneous abscesses and skin wounds. It also causes impetigo
(small pustules that form yellow crusty sores, usually around the
mouth). Penicillin and methicillin resistant strains of S. aureus
(MRSA) are common causes of hospital-acquired wound
infections.
 Severe invasive group A streptococcal infections with the toxic
shock like syndrome often begin with a simple skin or wound
infection.
 Pseudomonas aeruginosa is associated with infected burns and
hospital-acquired infections.
 Enterobacteriacae (e.g Escherichia coli, Proteus species ) are the
pathogens most frequently isolated from abdominal abscesses and
wounds.
 Gas gangrene can develop within a few hours of traumatic injury
and lead to rapid death. Clostridium perfringens is the most
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 common cause, and its -toxin produces the spreading tissue
damage and muscle death. It is found mainly in deep wounds
where anaerobic conditions exist and cause gas gangrene.

Microbial Culture

Once a microbiology laboratory receives bacterial specimens, it usually

has to perform two procedures known as culture and sensitivity tests.
Both rely on well-isolated colonies. The culture refers to the process of
growing the microbe on media appropriate for identifying the etiologic or
causative agent of the disease. The sensitivity procedure determines
which drugs the microbe is sensitive to.
Culture is microbial growth in a nutritional solid or liquid medium which
increased numbers of organisms. The bacteria grow into colonies, and the
colonies are counted. The culture of pathogens enables colonies of pure
growth to be isolated for identification and, when required, to test the
susceptibility of pathogens to antimicrobial agents.

Types of Microbiological Culture Media

For a culture medium to be successful in growing the pathogen it must

provide all essential nutrients, ions, and moisture, maintain the correct pH
and osmotic pressure, and incubated in the correct atmosphere, at the
optimum temperature and for an adequate period.

Culture media can be classified by consistency to solid, semi-solid and

fluid media (The major difference among these media is that solid and
semisolid media contain a solidifying agent (usually agar), whereas a
liquid medium does not) ; or by their content as basic media (nutrient
agar and nutrient broth), enriched media ( blood agar ), selective media
(Mannitol salt agar, TCBS), Indicator (differential) media (MacConkey
agar), Transport media (Cary-Blair medium) , Identification media
(peptone water sugars, urea broth, and Kligler’s iron agar).

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Choice of culture media depend on:
 The major pathogens to be isolated.
 Whether the specimens being cultured are from sterile sites or from
sites having a normal microbial flora.
 Cost, availability, and stability of different media.

Cultivation methods of microorganisms on culture media

1) Pour plate technique
2) Spread plate technique
3) Streak plate technique
The technique used to inoculate media in petri dishes (plates) must
provide single colonies for identification. It must also show whether a
culture is pure or mixed. A pathogen must be isolated in pure culture
before it can be identified and tested for antimicrobial sensitivity.

Antimicrobial Susceptibility Testing (Antibiogram)

Antimicrobial susceptibility testing methods are divided into types based

on the principle applied in each system. They include:
Diffusion : stokes method and Kirby-Bauer method
Dilution : minimum inhibitory concentration(MIC) which includes broth
dilution and agar dilution.
Diffusion and dilution : E-test method
Aim of this test :
• To measure susceptibility of an isolate to range of antibiotics.
• To assess emerging bacterial resistance patterns
There are some precautions must be taken during carrying this test
1) pH: ( 7.2 - 7.4).
2) Moisture: plates should be placed in an incubator (35C) or a laminar
flow hood until excess surface moisture is lost by evaporation .
3) Inoculum density: compared to 0.5 McFarland standard
4) Timing of disc application: 3–5 minutes (no longer than 15 minutes)
5) Temperature of incubation: 35 °C for optimal growth.
6) Incubation time: 16 - 18 hours.
7) Depth of agar media: not very thin or thick agar media.

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8) Spacing of the antibiotics discs: 25 mm apart and 15 mm from the
edge of the plate.
9) Potency of the antibiotic discs: not reduced or expired.
Disc diffusion methods
The Kirby-Bauer methods are usually used for antimicrobial
susceptibility testing.
Procedure
Inoculate the bacteria to be tested by homogenous streaking across the
agar plate. Apply the chosen antibiotics containing special concentration
of the drug at adequate spacing to the surface of the plate with sterile
forceps or disc dispenser (35-37 degree for 16-18 hours).
Measure the diameter of the zones of inhibition of bacterial growth.
Determine whether the bacteria is sensitive, moderately sensitive or
resistant to a specific antibiotic.

Collection of Microbiological Specimens

The value and reliability of microbiological reports are directly affected
by the quality of the specimen received by the laboratory and the length
of time between its collection and processing (the right one, collected at
the right time, transported in the right way to the right laboratory).
The laboratory should issue written instructions to all those responsible
for the collection of specimens from inpatients and outpatients
Basic instructions for specimens collection
Type of specimen
The correct type of specimen to collect will depend on the pathogens to
be isolated, e.g. a cervical not a vaginal swab is required for the most
successful isolation of N. gonorrhoeae from a woman.
Time of collection
Specimens such as urine and sputum are best collected soon after a
patient wakes when organisms have had the opportunity to multiply over
several hours. Blood for culture is usually best collected when a patient’s
temperature begins to rise.
Collection techniques
 Use a collection technique that will ensure a specimen contains
only those organisms from the site where it was collected.

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  If contaminating organisms are introduced into a specimen during
its collection or subsequent handling, this may lead to difficulties
in interpreting cultures and delays in issuing reports.
 A strictly sterile (aseptic) procedure is essential when collecting
from sites that are normally sterile, e.g. blood, CSF, or effusions.
 An aseptic technique is necessary not only to prevent
contamination of the specimen but also to protect the patient.
 Avoid contaminating discharges or ulcer material with skin
commensals.
 The swabs used to collect the specimens must be sterile and the
absorbent cotton-wool from which the swabs are made must be
free from antibacterial substances.
 Collect specimens in sterile, easy to open, leak-proof, dry
containers, free from all traces of disinfectant.
 Containers must be clean but need not be sterile for the collection
of feces and sputum.
 The containers given to patients must be easy for them to use.
 Patients should be instructed in the aseptic collection of specimens
and asked to avoid contaminating the outside of containers.
 When contamination occurs, wipe the outside of the container with
a tissue or cloth soaked in disinfectant before sending the specimen
to the laboratory.
 Report any abnormal features, such as cloudiness in a specimen
which should appear clear, abnormal coloration, or the presence of
pus, blood, mucus, or parasites.
 The appearance of urine, pus, vaginal discharge, feces, effusions,
and cerebrospinal fluid should be described routinely.
Labeling specimens
Each specimen must be clearly labelled with the date and time of
collection, and the patient’s name, number, ward or health center.
Specimens containing dangerous pathogens
Those delivering, receiving, and examining specimens must be informed
when a specimen is likely to contain highly infectious organisms. Such a
specimen should be labeled HIGH RISK.
Specimens which should be marked as HIGH RISK include:
 Sputum likely to contain M. tuberculosis.
 Fecal specimen that may contain V. cholerae or S. Typhi.
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  Fluid from ulcers or pustules that may contain anthrax bacilli or
treponemes.
 Specimens from patients with suspected HIV infection, viral
hepatitis, viral haemorrhagic fever, or plague.

Preservatives and transport media for microbiological specimens

In general, specimens for microbiological investigations should be
delivered to the laboratory without delay and processed as soon as
possible. This will help to avoid the overgrowth of commensals. When a
delay in delivery is unavoidable, a suitable chemical preservative or
transport culture medium must be used. This will help to prevent
organisms from dying due to enzyme action, change of pH, or lack of
essential nutrients.
A transport medium is needed to preserve anaerobes. Amies transport
medium is widely used and effective in ensuring the survival of
pathogens in specimens collected on swabs, especially delicate organisms
such as Neisseria gonorrhoeae. An example of a preservative is boric
acid which may be added to urine. Cary-Blair medium is used as a
transport medium for feces that may contain Salmonella, Shigella,
Campylobacter or Vibrio species.
Criteria for rejection of specimens
Events which may lead to the rejection of a specimen include:
 Specimen improperly labeled or unlabeled.
 Specimen improperly collected.
 Hemolysis, lipemia.
 Specimen sample volume not sufficient for requirement of test
protocol.
 Outside of container contaminated by specimen or patient not
properly prepared for test.
 All specimens must meet the requirements before being accepted
by the Laboratory.

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