Wound Infection

• Infection of soft tissues associated with pus formation
• Pus consists PMNs, bacteria, fibrin and other body fluids
• May be endogenous or exogenous

Endogenous: source of infection commensal flora, for example, abdominal surgical wound infection from normal flora of bowel

Exogenous:  source of infection outside the body, for example burn infection

Possible pathogens

• most commonly isolated pathogens from wounds, abscesses, burns, and draining sinuses.

BACTERIA

  Gram positive                                      Gram negative

S aureus                                             Pseudomonas aeruginosa

  S pyogenes                                                  Proteus spp

  Enterococcus spp                                             E. coli

Anaerobic streptococci                          Bacteriodes spp

Other streptococci                                    Klebsiella spp

  Clostridium perfringens                            Pasteurella spp

• FUNGI

Histoplasma

  Candida albicans

ULCER MATERIAL AND SKIN SPECIMENS

Gram positive                              Gram negative

Staphylococcus aureus                 E coli

  Streptococcus pyogenes                Proteus

  Enterococcus spp                     Pseudomonas aeruginosa

Anaerobic streptococci                       Yersinia pestis

Erysipelothrix spp                         Vincent’s organisms

BONE INFECTION

S aureus ,  H. influenzae

Group A Streptococci  Coliform bacteria

M tuberculosis (Bone TB)

CONS

Collection and transportation of sample

• should be collected by a medical officer or an experienced nurse

Collection time: best collected when abscess is incised and drained, or when wound ruptured naturally

Special precaution: should be collected before an antiseptic dressing is applied.

Procedure:

1.Using sterile needle aspirate from a drainage in to a tube up to 5 ml of pus.

2.Transfer to a leak-proof sterile container.

When pus not discharged

1.Use a sterile cotton-wool swab to collect a sample from the infected site.

(Sample may get desiccated and bacteria may become trapped in swabs)

2. Immerse the swab in a container of Amies transport medium

• If possible two swabs are taken, one for microscopy, next for culture
• If it can be arranged, use cooked meet broth, inoculate swab directly into broth immediately after collection

Transportation

• Without delay transport to lab and should be processed immediately

Routine investigation:

Examination for pyogenic and anaerobic pathogens

Special request for:
mycobacteria, actinomycetes , diphtheria , anthrax bacillus and fungi

• Pus from Actinomycetes infection contain granules, Small yellow granules

Macroscopic examination:

Appearance of pus  Presumptive organism

Creamy and thick                                  Staphylococcus aureus

Straw colored Watery                          Streptococcus pyogens

Fishy smell                                            Proteus spp

Blue pigmentation, musty odor            Pseudomonas spp

Putrid offensive smell                            Anaerobic infection

Actinomycetes                             Yellow colored granules

Fungal infection                               Black or brown granules

Microscopic examinations

Gram staining

• Gram positive cocci could be S. aureus or streptococci
• Gram negative rods that could be Proteus species, E. coli or other coliforms, P. aeruginosa
• Gram positive large rods with square ends could be C. perfringens or B. anthracis
• Large numbers of pleomorphic bacteria (streptococci, Gram positive and Gram negative rods of various size and fusiform bacteria), associated with anaerobic infections.
• Gram positive yeast cells with pseudohyphae , suggestive of Candida albicans
• Vincent’s organisms, if tropical ulcer is suspected, Gram negative spirochetes (Borrelia vincenti ) and Gram negative fusiform rods
  • Ziehl-Neelsen smear when tuberculosis or M. ulcerans disease is suspected
  • Potassium hydroxide preparation when ringworm or other superficial fungal infection is suspected
• CULTURE
  • inoculate on MA, BA, Cooked meat medium or thioglycolate broth

  Blood agar to isolate S. aureus and streptococci.

  MacConkey agar to isolate Gram negative rods.

  On Cooked meat medium to isolate anaerobs at the bottom of   tube

  • Incubate blood agar plate at 35–37°C in a carbon dioxide atmosphere (candle jar) one aerobic next anaerobic incubation.
  • MacConkey agar plate aerobically.
  • Cooked meat medium at 35–37 °C for up to 72 hours. Subculture at 24 h, and if indicated at 48 h and 72 h
• Anaerobic culture
  • Suspected anaerobic infection, specimen is often foul-smelling, or the Gram smear shows an ‘anaerobic mixed flora’, inoculate a second blood agar and incubate it anaerobically for up to 48 hours.
  • plate may be made selective by adding neomycin to final concentration of 50–70 g/ml
  • Majority of facultative anaerobic Gram negative rods will be inhibited.
  • metronidazole disc (5 μg) may be added to the anerobic blood plates, majority of anaerobes show a zone of inhibition, whereas aerobes grow up to the disc

  DAY 2

  Examine for relative number and types of colonies

  If no colonies are isolated, report no growth

• FEW CONSIDERATIONS
  • Pure growth of recognized pathogens is reported as significant
  • Scanty growth of skin commensals like CONS, usually disregarded and not reported
  • Few colonies of E. coli potentially isolated from wound likely to contaminated with fecal bacteria is considered as not significant
  • Pure growth of commensal type organisms isolated from deep site aspirate considered significant

  IDENTIFICATION OF ISOLATES

  Gram stain, Coagulase for Stahylococci

  Lancefield grouping of beta hemolytic streptococci

  Biochemical test for coliforms and anaerobs

• Anaerobic blood agar culture and cooked meat culture
  1. perfringens:

  Cooked meat medium: Grows rapidly in with H2S production (gas bubbles in turbid medium) and reddening but no decomposition of the meat.

  Anaerobic blood agar: colonies are usually seen after 48 h incubation, most strains produce a double zone of haemolysis (inner zone of clear haemolysis, outer zone of partial haemolysis.

  1. fragilis:

  CMM: decomposition of meat with blackening of the meat (foul-smelling proteolytic reaction).

  Anaerobic blood agar: non-haemolytic grey colonies

• PeptostreptococcusCooked meat medium: production of large amounts of hydrogen sulphide gas.

  Anaerobic blood agar: small nonhaemolytic white colonies

  How to confirm organism are anaerobic?

  When there is a mixed growth and colonies appear on the anaerobic plate and not present on the aerobic plate, confirm that the organisms are anaerobes by sub inoculating the colonies on three plates of blood agar and incubating one aerobically, one anaerobically, and the third in a carbon dioxide atmosphere (candle jar).