Dr. Kiarash Ghazvini Department for bacteriology and virology, Mashhad University of medical Sciences

2. Dr. Kiarash Ghazvini Department for bacteriology and virology, Mashhad University of medical Sciences Wound Infection

3. WOUNDS AND ABSCESS • Infections of soft tissues are associated with production of pus and said pyogenic. • A wide variety aerobic and anaerobic species participate singly or in combination in infectious of wounds.

4. WOUNDS AND ABSCESS • The commonest pyogenic bacteria areS.aureus, S .pyogenes ,P. aeruginosa, coliforms bacilli., anaerobic organisem :particularly Clostridium perfringens , bacteroides spp ,anaerobic cocci.. • In many cases there is a mixed infection with more than one bacterial spp.

5. wounds Wound infections may be endogenous or exogenous. Endogenous infections are caused by organisms that are in commensal in the patient . Exogenous infections the source is out of the body cross-infection is a particular example.the causal organism is spread from person to person. infection may occur after accidental or intentional trauma of the skin or tissue. (Surgical postoperative sepsis. )

6. Wounds are liable to contamination with a multiplicity of organisms from the body surfaces and environment. • Infection of a wound is difficult to define , and no clear rules can be given to distinguish it from colonization and contamination.

7. Contamination • The contaminating organisms are present in small numbers

8. Colonization • In the case of commensal or low grade pathogenes it can be described colonization.

9. Infection • Infection occurs when they evades the host defences.replicates in large numbers and attacks the host tissue.

10. Acute Caused by external damage to intact skin Types Surgical Bites Burns Minor cuts Abrasions Severe traumatic Chronic Precipitated by predisposing conditions that lead to compromise of dermal/epidermal tissue Types Impaired venous drainage Impaired arterial supply Metabolic diseases eg. diabetes Wounds:Classification

11. Wound infections: Etiology • Surgical wounds • Aerobes: S. aureus, coagulase negative staphylococci, Enterococcus spp. E. coli, P. aeruginosa, Enterobacter spp. • Anaerobes: Bacteroides spp., Peptostreptococcus, Closthdium spp. • Acute soft tissue infections • Staph aureus only organism in 30% • 30-50% mixed aerobes/anaerobes • 20-30% other eg. Group A streptococci, Clostridium spp. • Bite wounds • Special pathogens: Pasteurella muftocida, Capnocytophaga canimorsus, Bartonella henselae, Eikenelfa corrodens • Other mixed aerobes and anaerobes

12. Wound infections: Etiology • Burn wounds • Primarily aerobic organisms: P. aeruginosa, Staphylococcus aureus, E. coli, Klebsiella spp. Enterococcus spp. and Candida spp. • Diabetic foot ulcers • Aerobes: Staph aureus, Streptococcus spp. P. aeruginosa, Enterococcus spp., enterics • Anaerobes: Peptostreptococcus, Bacteroides spp., Prevotella spp. • Decubitus ulcers • Mixed aerobic and anaerobic bacteria

13. SAMPLING • Biopsy ● Aspirate from depth of the wound • if possible pus or exudate should be submitted in a small screw-capped bottle, a firmly stoppered tube or sterile syringe and needle.

14. SAMPLING • Swabs are inefficient sampling and are strongly discouraged. • If it is decided to send swabs tow swab is necessary , one for microscopy ,one for culture.

15. SAMPLING • Delay in the transit of specimen to the laboratory must be avoided.especially swabs where the exudate may dry.

16. CONTINUE… • If the swab is dry, moisture it well with a little sterile broth or saline . • the examination of material on swabs for mycobacteria is always unsatisfactory. • Physicians should be instructed that when a special investigation is required ,they usually should state on the request form.

17. SAMPLING ● Clean the wound margins with 70% ethyl alcohol.

18. Wound Cultures • For closed Wounds • Prepared site as described for obtaining Blood Culture • Aspirate as much purulant material as possible • Transport in aerobic /anaerobic transport media

19. Laboratory examination • Special methods of examination should be applied to particular specimens. • the basic procedures usually include • Naked eye examination, (for macroscopy criteria ,such as color , odor consistency,..) • The microscopical examination, • Culture on aerobic and anaerobic blood agar plates, on MacConkey agar and in cooked –meat broth .

20. Microscopy • Much useful information may be obtained from smear by Gram-staining. We should notice • presence and relative numbers of PMNs , SEC and bacteria • morphology of Gram – positive or Gram – negative bacteria. • Examination of a wet film for fungi or motile bacteria. • A smear stained by the Ziehl- Neelsen method should be examined when the clinical circumstances suggest the tubercle bacillus., another mycobacterium or a nocardia may be present

21. Wound Cultures • Culture for aerobic and anaerobic bacteria if appropriately collected • Gram stain results suggest adequate collection or presence of inflammation • Tissues or aspirates vs. swabs • Primary plating media: 5% SBA, Choc agar, MacConkey agar; anaerobic plates and thio if appropriately collected • Identify anaerobes to Genus level only • Perform susceptibility testing of predominant organisms only

22. CULTURE • the specimen should be inoculated on two plates of blood agar (SBA 5%), • the one for incubation at 37c, 5-10% CO2.,for 18-24h. • The other for incubation anaerobically . • It should also be plated on Mac Conkey or CLED agar, for identification of coliforms , staphylococci ,enterococci, also be inoculated into a tube of cooked –meat broth for the enrichment of exacting aerobes and anaerobes.

23. CULTURE • Colonies should be noted and more tests for identification and antibiotic susceptibility tests done. • If there is no growth after 24h ,all plates should be reincubated for another 24h • for slow-growing pathogen such as Actinomyces israeli or some species of bacteroides it should be incubated longer for about 7 days.

24. CULTURE • if at 24 h or 48 h there is growth on cooked-meat broth , but no growth on the plates , the broth should be filmed and subcultured. • if tuberculous or fungal infection is suspected , the specimen should be cultured by the appropriate methods on special media.

25. Wound Cultures: Extent of Workup Possible approaches • Use Gram stain result • Work up organisms seen on stain only • List others • Work up any potential pathogens to maximum of three, list others present by morphology • Work up any quantity S. aureus, P. aeruginosa, beta hemolytic streptococci, enterics and gram-negative anaerobes

26. Wound Specimens: Algorithms • Three approaches • PMN predominance • Q-Score • Q-2-3-4 system

28. Wound Cultures: Examples • Gram stain results: (Acceptable) • Many neutrophils, no epithelial cells, Many gram positive cocci in clusters, Many gram negative bacilli, Few morphotypes resembling skin flora • Work up (identify and perform susceptibility testing): Gram positive cocci in clusters and gram neg bacilli • Culture report: Many S. aureus, many Klebsiella pneumoniae, light aerobic bacteria resembling skin flora

29. Workup of Wound Cultures • Q-Score System • Good quality specimen (Q3) • Up to 3 organisms can be considered as potential pathogens and worked up (ID/AST) • Lower quality specimen (Q2, Q1) • More SEC • Fewer organisms are worked up

30. Workup of Wound Cultures • Q-Score System • If the Q-score is greater than or equals the PP in culture • Workup all potential pathogens • If Q-Score is less than the PP in culture • Look at the Gram stainWorkup all PP that are seen on GSMorphologically ID othersIf all PP present on GS then only Morph ID all

32. Wound Cultures: Examples • Gram stain: many neutrophils, few epithelial cells, Gram positive cocci in clusters, Gram positive cocci in chains, • Culture grows: many S. aureus, many Group A streptococci, few enteric bacilli • Work up: S. aureus, Group A streptococcus: limited ID and no susceptibility on enteric bacilli susceptibility testing on Group A strep not required

33. Wound Cultures: Example • Gram stain: many neutrophils, few epithelial cells, Gram positive cocci in clusters, Gram positive cocci in chains, • Culture grows: many S. aureus, many Group A streptococci, few enteric bacilli

34. Workup of Wound Cultures • Q/2-3-4 System • Culture workup is based on the # of PP present • 2PP-ID/AST • 3PP • Look at the Gram stain • " Workup two PP if they are seen on GS • " If all 3 present on GS then Morph ID • 4PP • Morph ID only

35. Wound Cultures: Example • Q score = 2 [PMN (+3), few epi (-1)] • Q/2-3-4 = 3 PP • look at gram stain • Work up: S. aureus, Group A streptococcus, Morph ID and no susceptibility on enteric bacilli

36. Interpretation and reporting • A pure growth of a recognized pathogen obtained from a wound or closed abscess is easily interpreted as significant and will be reported to the physician as being so. • Mixed cultures grown from superficial lesions are the basic difficulty.

37. Interpretation and reporting • Scanty growths of skin commensals such as staph or diphteheroid bacilliare usually disregarded and not reported.and a few colonies of E.coli grown from a perineal . But clostridium perfringens is important. • In superficial lesions such as varicose ulcers , present of mixed commensal is not important. • The result is reported: Many mixed fecal and skin bacteria present. without giving identities or antibiotic sensivities.

38. But a pure growth of a commensal from an aspirated deep wound is not contamination and should be reported with AST performance. • In general, a numerous or predominant organism is likely to have pathogenic significance.

39. But the relative numbers of the colonies of the different organisms on a culture plate may not reflect the relative numbers of the organisms in the lesion .for they are subject to many variations such as the relative speed of growth of different species., antibiotic interactions between different speciesand the greater tendency of the more delicate pathogenes to die during transport of specimens. • For such reason a causal pathogen may be cultured in smaller numbers than a contaminating commensal.

40. Wound Cultures: Examples • Gram stain: Many neutrophils, few epithelial cells, multiple morphotypes • Culture grows: more than 3 potential pathogens • Consider source • Tissue or aspirate ? • Contamination likely ? • Type of patient • May need to consult with clinician or Infectious Diseases service