SPECIMEN ANALYSIS:ABSCESS ASPIRATEAnneka Pierzga and Yackima Saura-WelchJune 16th, 2016MLT 2010 Clinical Microbiology (Professor Tiffany Gill) – College of Southern Maryland

THE SPECIEMN What is an abscess?

Accumulation of purulent material in the dermis or subcutaneous tissue

Appears as a swollen, red, tender and fluctuant mass Often diagnosed based on history and physical

exam, although studies suggest that soft-tissue ultrasonography may enhance accuracy of abscess detection, especially in cases of deeper or questionably appearing abscesses

Culture of needle-aspirated material useful for isolation of causative pathogens

SPECIMEN COLLECTION1. Cleanse site with sterile saline

or 70% alcohol2. Aspirate area containing

purulent material or fluid by needle and syringe (may need to irrigate with a small volume of non-bacteriostatic sterile saline)

3. Expel aspirated material in to sterile screw top tube

4. For anaerobic culture: Samples should be placed into oxygen free environment using anaerobic transport media

SPECIMEN COLLECTION

SPECIMEN COLLECTION

SPECIMEN TRANSPORT

SPECIMEN TRANSPORT “BD Port-A-Cul™ tubes, jars and vials contain a reduced transport

medium and are intended to maintain the viability of anaerobic, facultative and aerobic microorganisms during transport from the patient to the laboratory. Sterile packages are for collection of specimens in clean areas; e.g., surgical suites.”

PRIMARY SET-UP

PRIMARY SET-UP Direct examination of a Gram Stained slide

Determine the staining and morphological characteristics of pathogens to direct physician’s initial treatment plan

Inoculation of media Blood Agar Plate Chocolate Agar Plate MacConkey Agar Plate CNA Anaerobic (Columbia Agar with Colistin and Nalidixic

Acid) BBA (Brucella Blood Agar) LKV (Laked Blood Agar with Kanaycin and Vancomycin) BBE (Bacteroides Bile Esculin Agar)

CAUSES OF ABSCESSES

MAJOR OFFENDERS The leading cause of subcutaneous

abscesses in otherwise healthy individuals is…

Staphylococcus aureus Methicillin-Resistant Staphylococcus

aureus has been found to be the most common cause of abscesses in patients presenting to the emergency department in the US, followed by methicillin-susceptible S. aureus and beta-hemolytic streptococci

About Staphyloccos aureus:• Virulence factors• Colony morphology• Hemolysis• Environmental conditions• In vitro contamination• Common normal flora• Differential media• Confirmatory testing• Antibiotic susceptibility testing

Staphyloccos aureus Gram-positive cocci in grape-like clusters

Found as part of the normal flora of the anterior nares, nasopharynx, perineal area, skin, and mucosa; may be introduced to sterile sites by traumatic introduction

May also be spread from person to person by direct contact

VIRULENCE FACTORS

Polysaccharide capsule inhibits phagocytosis and helps with colonizationCatalase helps resist digestion by leukocytesPenicillinase provides resistance to penicillin-related antibioticsCoagulase enables the bacteria to hide within a clot, thereby escaping the complement system

S. aureus COLONIES

S. Aureus colonies typically appear to cream in color but occasionally have a yellow pigment.

The golden pigmentation (staphyloxanthin) has been reported to be a virulence factor protecting the pathogen against oxidants produced by the immune system

To compare in size and color S. epidermidis appears white.

Medium to large (0.5-1.5 μm); smooth, entire, slightly raised, low convex, opaque; most colonies pigmented creamy yellow; most colonies beta-hemolytic

HEMOLYSIS

Beta-hemolysis (β-hemolysis), sometimes called complete hemolysis, is a complete lysis of red cells in the media around and under the colonies: the area appears lightened (yellow) and transparent. .

ENVIRONMENTAL CONDITIONS & INCUBATION PERIOD

Grows best aerobically but are facultative anaerobic.

24 hours of incubation at 37°C

IN-VITRO CONTAMINATION

Meaning: When something foreign and non-sterile has made contact with the plate during inoculation.

Can occur by Not cleaning collection site

appropriately Not streaking plate under

the hood Opening the lid and

breathing/allowing any micro-contaminant to enter.

Not using sterile inoculating loop

The arrows indicate fungal contamination of the specimen

NORMAL FLORASince many abscesses are located beneath the skin, it is not uncommon to have normal skin flora in the sample.Coagulase negative Staphylococcus species and Enterococcus species are considered normal skin flora

DIFFERENTIAL/SELECTIVE MEDIA

Phenylethyl alcohol agar (PEA) Mannitol Salt Agar (MSA) has a 7.5%

concentration of salt and S. aureus can ferment mannitol

Columbia colistin-nalidixic acid (CNA) agar Chromogenic media DNase or thermostable-endonuclease test.

CONFIRMATORY TEST

• Gram - positive cocci

in grapelike clusters• Catalase - positive• Coagulase - positive

SUSCEPTIBILITY TESTINGIF SUSCEPTIBLE: ampicillin/sulbactam amoxicillin/clavulanate oxacillin nafcillin cefazolin ceftriaxone Macrolides ClindamycinALTERNATIVES: Trimethoprim-Sulfomethoxazole(TMP-

SMX) vancomycinMRSA: vancomycin teicoplanin linezolid quinupristin/dalfopristin TMP-SMX rifampicin

Resistance to 0.04 U of bacitracin (Taxo A disk) and furazolidone

REFERENCESBacteria in Photos. (2016). Beta hemolysis of Staphylococcus aureus. Growth characteristics of staph aureus on blood agar. Retrieved from http://www.bacteriainphotos.com/beta_hemolysis_on_agar.htmlBD (Becton, D. a. C. (2016). Diagnostic Systems: Port-A-Cul™ Tube (for Aerobic and Anaerobic Cultures). Retrieved from http://www.bd.com/ds/productCenter/221606.aspCardiovascular and Interventional Radiological Society of Europe. (2016). Aspiration.Microbiology in Pictures. (2016). Staphylococcus aureus colony morphology and microscopic appearance, basic characterisic and tests or identiication of S. aureus bacteria. Retrieved from http://www.microbiologyinpictures.com/staphylococcus%20aureus.htmlSankaqm5 (Producer). (2012, 6/11/2016). Large Abscess Cavity Pus Aspiration With Anti Gravity Technique Retrieved from https://www.youtube.com/watch?v=xP1-ccR4O2wSinger, A. J., & Talan, D. A. (2014). Management of Skin Abscesses in the Era of Methicillin-Resistant Staphylococcus aureus. The New England Journal of Medicine, 370(11), 1039-1047. Talan, D. A., Krishnadasan, A., Gorwitz, R. J., Fosheim, G. E., Limbago, B., Albrecht, V., & Moran, G. J. (2011). Comparison of Staphylococcus aureus From Skin and Soft-Tissue Infections in US Emergency Department Patients, 2004 and 2008. Clinical Infectious Disease, 53(2). The University of Texas Medical Branch. (2016). Specimen Collection: Acceptable Specimens for Bacteriologic Analysis of Wounds. Retrieved from https://www.utmb.edu/lsg/Pages/SpecimenCollection/SpecColWounds.aspxTille, P. M. (2014). Bailey and Scott's Diagnostic Microbiology (13 ed.): Elsevier.