20/02/2013. Blood Cultures How they work Key pathogens Interest and value. Blood cultures:

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1 Blood Cultures How they work Key pathogens Interest and value Jan Jacobs, MD, PhD, Professor in Tropical Medical Biology Institute of Tropical Medicine, Antwerp, Belgium Blood cultures: Blood is sterile: there are no bacteria in the blood! Bacteria that enter the blood are killed by white blood cells Bacteremia = presence of bacteria in the blood Septicemia = bacteremia + clinical signs/symptoms (fever, shock ) mortality 20 40% Blood culture = culture of bacteria from the blood 1

2 Outlines of this presentation 1. Refresher of Bacteriology 2. How do bacteria get into the blood? 3. Blood cultures: how they work 4. Blood cultures: bacteria that we expect 5. Blood cultures: interest (over other cultures) Helminths (Worms) (some) are visible by naked eye multicellular Protozoa need microscope 2

3 Fungi Mold Yeast Mold 0.002µm 0.2µm 0.2mm Electron mic. Optical mic. Naked eye Protozoa 25µm - 5µm Yeast 5µm Bacteria HIV HB virus 2µm 0.2µm 0.1µm 0.04µm 3

4 Cell wall = Inner membrane Peptidoglycan Grampositive Cell wall = Inner membrane Peptidoglycan Outer membrane Gramnegative A short course on bacteriology Grampositive Gramnegative Rod Examples? Coccus 4

5 Grampositive Gramnegative Rod Coccus Gram positif Gram négatif Bacilles Clostridium tetani Salmonella typhi Coques Staphylococcus Spores aureus Méningocoques Sec = Spore Clostridium Bacillus 5

6 Gram-positive Staphylococcus aureus Cocci clusters Streptococci Cocci chains Streptococcus pneumoniae Cocci (lancet), duplo, capsule Gram-negative Haemophilus Cocco-bacilli Nesseria Cocci duplo Escherichia coli Klebsiella Salmonella Enterobacteriaceae Pseudomonas, Burkholderia Non-fermentors Corynebacteria Fine rods Bacillus, Clostridium Rod, spores Vibrio cholerae Rod comma Campylobacter Rod corckscrew White blood cells with Grampositive cocci in clusters Gramnegative rods (with capsule) in blood culture 6

7 Grampositive cocci in (short) chains Gramnegative curved bacteria (Campylobacter) Grampositive rods and Grampositive cocci 7

8 Grampositive rods (partially discolored) with spores (Clostridium, Bacillus) Grampositive rods, spider-like Propionibacterium spp. 8

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10 Bacteria Metabolism Aerobe: Anaerobe needs oxygen Pseudomonas aeruginosa, Pseudomonas aeruginosa does not tolerate oxygen! Clostridium perfringens Facultative aerobe/anaerobe can grow with and without oxygen Escherichia coli Capnophilic needs some additional CO 2 Streptococcus pneumoniae Gram positive Gram negative Rod Clostridium tetani Escherichia coli Anaerobic Aerobic/Anaerobic Cocci Staphylococcus aureus Aerobic/Anaerobic Meningococcus Aerobic Aerobic = needs oxygen Anaerobic = does not tolerate oxygen Growth of Bacteria: what is needed? Much water Some carbodydrates (sugars) Some nitrogen (amino acids) Some salts Some vitamines and minerals Temperature ph 10

11 How do we culture bacteria? Sugar + Water +++ Proteins + Vitamins and minerals ph temperature Bacterial flora: Environment High numbers Low numbers Warm Moist Dry High sugar concentration High salt concentration Acid/Alcaline 11

12 Bacteriology - reminder Gram positive Gram negative Rod Cocci Outlines of this presentation 1. Refresher of Bacteriology 2. How do bacteria get into the blood? 3. Blood cultures: how they work 4. Blood cultures: bacteria that we expect 5. Blood cultures: interest (over other cultures) How do bacteria reach the bloodstream? 1. Infections in organs 2. Endovascular Infections 3. Abscess 4. Generalised Infections 12

13 Infections of organs Lung Pneumonia: Streptoccoccus pneumoniae, pneumococcus Kidney Escherichia coli Pyelonephritis: Bone Joints: Osteomyelitis, arthritis: Staphylococcus aureus Abscess e.g. abdominal abscess Gramnegative rods Enterocococci 13

14 Endovascular Infections Generalised Infections Beta-hemolytic streptococcus Typhoid fever 14

15 How do we detect bacteria in the blood? Urinary tract infection Pneumonia : bacteria/ml : bacteria/g Bacteremia : 1 10 bacteria/ml (adult) bacteria/ml (child) 1% of red blood cells = /ml Outlines of this presentation 1. Refresher of Bacteriology 2. How do bacteria get into the blood? 3. Blood cultures: how they work 4. Blood cultures: bacteria that we expect 5. Blood cultures: interest (over other cultures) 15

16 How do we culture bacteria? Blood culture bottle Brain Heart Infusion (BHI) broth Sugar + Water +++ Protein + Salt Vitamins, minerals ph Sodium Polyanethol Sulfonate (SPS) anticoagulant, inhibits phagocytosis, neutralises antibiotics Manual System: 1 set = 1 Aerobic bottle + 1 Anaerobic bottle Ratio 1/5-1/10 Dilution antibodies & antibiotics 50 ( 100) ml of Broth 5 ( 10) ml of Blood 16

17 Analysis in the laboratory Check for Growth - Daily Observation during 7 days - 10 to 15% of Cultures will grow - Most bacteria grow within 3 days - Gram stain - Report by phone call Subculture on agar plates Which bacterium causes the disease? Antimicrobial resistance Which antibiotics can we use? What conditions are needed for blood cultures 1. Volume of blood drawn 2. Moment of Collection 3. Aerobic/Anaerobic bottles Volume of blood sampled (adults) % growth ml 20 ml 30-40ml 1 set = 1 venipuncture, 2 sets = 2 venipuncture 17

18 Moment of Collection Tooth extraction (transitory) Abscess (intermittent) Endocarditis (continuous) Aerobic/anaerobic bottle 1. Aerobic bottle is the most important: - Fill this bottle first 2. Additional information of the anaerobic bottle is limited - Strictly anaerobic bacteria = rare - Clinical picture of anaerobic infections usually suggestive (abdominal, genital, gangrene) - No resistance Limited resources: do not use anaerobic bottle Collection: recommendations Manual System 3 sets with a delay of 60min (3 x 2 x 5 to 10 ml) 1 set = 1 puncture 1 set = 1 aerobic + 1 anaerobic bottle Feasibility/costs: 2 punctures 10ml/puncture 1 bottle/puncture At the moment of chills/fever 3 sets after 24h if symptoms are persisting Before start antibiotics (or before next administration) 18

19 Collection, children Manual systems Automatic sytems 2 5 ml 3ml, 4 ml Children = 0 - < 14 years old Neonate: ml Collection of Blood Cultures Cambodia Adults 2 bottles 10 ml in each bottle Children < 1 year: ml 1 year - < 2 year: 2 ml 2 year - <14 year: 5 ml Outlines of this presentation 1. Refresher of Bacteriology 2. How do bacteria get into the blood? 3. Blood cultures: how they work 4. Blood cultures: bacteria that we expect 5. Blood cultures: interest (over other cultures) 19

20 Bloodstream infections in SHCH: distribution over Clinically important bacteria seen in blood cultures in Cambodia Aerobes Gram positive Gram negative Rods: Corynebacterium Listeria Actinomyces Lactobacillus Erysipelothrix Bacillus Nocardia Mycobacterium (also acid fast) Cocci: Catalase positive: Staphylococcus Micrococcus Catalase negative: Streptococcus Enterococcus Rods: Enterobacteriaceae E. coli, Klebsiella, Proteus, Serratia, Morganella, Shigella, Salmonella, Yersinia, Enterobacteri, etc. Acinetobacter Pseudomonas Stenotrophomonas Burkholderia Aeromonas Vibrio Campylobacter Brucella Francisella Bordetella Eikenella Pasteurella Haemophilus Legionella Cocci: Neisseria Moraxella Key Tests used to Identify GNRs Colony growth & morphology on blood agar and MacConkey Utilization of Glucose Oxidase Oxidizer/nonoxidizer or fermenter 20

21 Enterobacteriaceae Gram negative bacilli; grow well on BAP & most species grow on MacConkey agar Oxidase negative Able to ferment glucose Most live in the patient (intestine) but survive in environment Urinary tract infections Abdominal infections Problem of antibiotic resistance ESBL (extended spectrum beta-lactamase) and carbapenemase) Ferment glucose in Triple Sugar Iron Agar (TSI) Salmonella Gram-negative rods, Enterobacteriaceae Salmonella Typhi, Salmonella Paratyphi A Enteric (typhoid) fever Generalised infection (gut perforation and peritonitis) Multidrug- and fluoroquinolone resistance Non-Typhi Salmonella Salmonella Choleraesuis, Salmonella Typhimurium, Enteritidis Multi-drug resistance In adults: indicator for HIV-infection Burkholderia pseudomallei Major pathogen in pneumonia, abscesses, skin lesions, blood cultures Catalase + Oxidase + Rough colony Strong musty smell Bipolar-stain Gram - rod B. cepacia: smooth, pale greenish colonies on BAP Rough, pink colonies on MacConkey Characteristic pseudo cepacia Pseudo. mallei stutzeri Wrinkly colony + Neg Later Polymyxin & R R R Gentamicin Amox/Clav S Most R R Musty odor + Neg Neg Ellen Jo Baron 2012; Use with proper attribution 21

22 Burkholderia pseudomallei Causes Melioidosis Gram-negative rod, does not ferment glucose No fermentation of glucose in Triple Sugar Iron Agar (TSI) Environmental organisms Diabetes, alchohol Serious sepsis and pneumonia (can look like TB) Abscesses Specific treatment (ceftazidime for systemic disease) High mortality Staphylococcus aureus Gram positive coccus, clusters Hemolytic toxin Skin and nose colonisation 1/3 of population) Skin and soft tissue infections Arthritis, osteomyelitis Bacteremia risk of endocarditis! (repeat blood cultures after 5 days) Abscesses by dissemination through blood MRSA (methicillin resistant Staphylococcus aureus) often multidrug-resistant especially resistant to Ciprofloxacin Streptococci After bile added Streptococcus pneumoniae Gram-positive cocci, diplo, lancet, capsule Dies at low temperature! Dies if grown out becomes Gram-negative Pneumonia, meningitis, otitis, sepsis, abscesses Streptococcus suis pneumococcus of the pig Optochin or bile resistant (simple test in lab) Meningitis + sepsis Middle-aged, women 22

23 Streptococci (other) Hemolytic streptococci Large colony, clear hemolysis Groups A, B, C and G Skin and soft tissue infections (respiratory or urinary tract) Viridans streptococci (Oral streptococci) Long chains No hemolysis (or alpha-hemolysis) Small colony, caramell-odor, Transient (children) may be hemolytic Endocarditis (adults) Streptococcus anginosus group (S. milleri): think abscess Enterococcus Short chains, diplo, bile-esculin resistant Rare in blood cultures Endocarditis, gastrointestinal infections (mixed infections) Non-fermentors Gram-negative rod, do not ferment glucose Mostly resistant to antibiotics and disinfectants Mostly from environment and most need water Major pathogens a. Pseudomonas: needs water (Pseudomonas aeruginosa, Xanthomonas.) b. Acinetobacter: resists dryness c. Burkholderia pseudomallei and cepacia: (need water/humidity, such as rice fields) Especially when occurring in clusters, think about external common source for infection (multidose vial, contamination of equipment..) Acinetobacter baumanni complex Can be a pathogen in any site Often multi drug resistant Gram or Gram variable coccobacilli. Tends to stain Gram positive in original specimens. Smooth round colonies on BAP Non-lactose fermenters (purple) on MacConkey Non- hemolytic Fish-like smell Oxidase negative Indole negative Colonies resemble Enterobacteriaceae on BAP but are blue-purple on MacConkey. Gram coccobacilli from colonies Ellen Jo Baron 2007; Use with proper attribution 23

24 Outlines of this presentation 1. Refresher of Bacteriology 2. How do bacteria get into the blood? 3. Blood cultures: how they work 4. Blood cultures: bacteria that we expect 5. Blood cultures: interest (over other cultures) Interest of blood cultures 1. Individual patient: correct diagnosis = better prognosis 2. Medical/Nursing team: insights in diagnosis 3. Surveillance: hospital - nationwide - which bacteria occur in a hospital/country? - which is their resistance? 4. Infection control : hospital acquired epidemics Standard treatment guidelines Surveillance reports, hospital (nosocomial) epidemics 24

25 Saliva: epithelial cell with commensal flora Normally sterile body fuids Cerebrospinal fluid Blood Lung (distal) Joint fluid Peritoneal fluid Pleural fluid Urine 1. Collection is easy and reliable 2. Blood cultures are more frequent and representative for the bacteria that cause disease in a population 25

26 Specimen types that may be contaminated with commensal flora Urine: Sputum: Ulcers, wound Blood: skin and intestinal flora oral flora 3. Urine/Sputum cultures are more selected (bias to resistance - S. aureus or Gram-negative bacteria stick to ulcers colonisation also/but less contamination by skin flora! (see quality indicators) 4. Blood culture less prone to contamination and contaminants are better recognised (skin flora) Two other reasond to chose for blood cultures Urine, Sputum, Wound: Collection and Transport Mixed flora 5. Blood cultures less demanding (for instance no cold chain) Sputum, Wound (Urine): More that one bacterium Mixed flora = difficult to workup 6. Blood cultures in > 95% only a single bacterium 26