Blood culture in infectious disease

diagnostic

Blood culture ◦ Cultivation of blood sample ◦ Want to isolate and identify the bacteria or

fungus from the blood, in some pathologic conditions.

Bacteriemia – the presence of bacteria in the blood for a short period of time, without clinical expression.

Septicaemia – systemic disease associated with the presence and persistence of the microorganisms or their toxins in the blood.

Definitions:

Normally the blood is sterile In blood culture can grow:

◦ Contaminants bacteria from skin, during sampling ◦ Microorganisms occasionally identified in the blood, in

different situation: Skin lesions (catheter, teeth extraction) Obstruction of billiary or urinary tract) Profound suppuration In most of cases, the episode are transitory. In case of

patients with endocard lesions, there is conditions for infectious endocarditis developing.

◦ Bacteria which are frequent isolated from: Infectious endocardytis; Enteric fever Infectious with Haemophilus influenzae (type b). Brucellosis, Frequent in acute lobar pneumonia, meningitis,can develop infections with septic metastasis localization.

Physiopathology

Enterobacteriaceae◦ Escherichia coli…………………..............................1751 cases◦ Klebsiella spp……………………………………..................765◦ Enterobacter spp………………….............................399◦ Serratia spp…………………………………………………………..136◦ Proteus mirabilis……………………………………………………122◦ Salmonella, all serotypes………………………………..93◦ Citrobacter spp……………………………………………………….76◦ complex Enterobacter agglomerans.…………………….44◦ Morganella morganii…………………………..................26

Other microorganisms◦ Staphylococcus aureus……………………………………….2151◦ Coagulase negative Stafilococci ……………………………1256◦ Enterococcus spp………………………………………………….794◦ Streptococcus pneumoniae………………………………...475◦ Pseudomonas aeruginosa……………………………………451◦ Streptococi beta-hemolitici…………………………………307◦ Acinetobacter spp……………………………………………….206◦ Streptococi viridans……………………………………………154◦ Stenotrophomonas maltophilia……………………………69◦ Haemophilus spp………………………………………………….27◦ Corynebacterium spp…………………………………………..20

Microorganisms isolated from patients with bacteremia \

◦ sterile transfer kit◦ culture media

system of manually manufacture and visual reading;

automatic systems; systems of centrifugation for mycobacterium

detection and other intracellular bacteria.

◦ piece of oil cloth 40/40 cm◦ solutions for decontamination:

liquid soap; iodine 2%; ether :

remove iodine; dry the skin .

Procedure – necessary:

Diphasic culture media

Pathologic product: blood. When?

Onset of disease; Before antibiotic administration During shiver

How much?◦ Adult: 3 samples x 20 ml, from different veins, at 30‘ interval◦ Children: 1 sample x 1 – 3 ml.

How ?◦ Skin washing ◦ Disinfection with iodine ◦ Skin drying with ether◦ Puncture of the vein with sterile nedle ◦ Introduction of the blood in culture media for blood culture: for every

sample are used 2 flacons: one with aerobic incubation and one with anaerobic incubation. The bottles are incubated in thermostat at 37 °C and are kept for 2 weeks.

When can we say that the blood culture of the patient is positive? - Solid phase: appearance of the colonies;- Liquid phase:

turbidity destruction / coagulation of erythrocytes appearance of gas. Next steps:

◦Microscopic examination of the colonies from the solid phase ◦Subculture◦Biochemical and antigenic identification ◦Antibiogram.

Procedure of blood culture

1. Sampling before antimicrobial therapy;2. Sampling from different veins, for each blood

culture;3. Avoidance of sampling through i.v. catheter 4. Quick transportation at laboratory, or, if it is not

possible, incubation of bottle at 37C, until the moment of sending.

5. Isolation of the same bacteria from many bottles.

Quality criteria for blood culture:

Clinical diagnosis Time of sampling In case of antimicrobial treatment, the

specification of the antibiotic that was administered

If the sampling was made during the high fever.

Analysis requesting – specifications:

The arguments of the microbiolog: Has clinical significance: isolation of the same bacteria

(species) in 2/3 vials and from different veins. ◦ Contamination: isolation of different bacteria from blood

culture bottles, from the same patient◦ Quantitative blood culture help to argue the clinical

significance of the conditioned pathogen; it is indicated when the skin decontamination is difficult.

◦ The presence of some bacterial structure involved in the pathogeny of catheter bactaeremia ( biofilms );

◦ The argumentation of poly-microbial bactaeremia is based on isolation of at least 2 microorganisms from the same blood culture, at least two times in 24 hours.

The arguments of the clinician: ◦ The age and the immune status of the patient;◦ The characteristics of the primary septic focus; ◦ Inflammatory system: leukocyte, CRP, fibrinogen.

Results interpretation

The absence of bacteria from the sample; Low sensitivity of the method; Samples collected after treatment with

antibiotics.

The analysis of the bacterial growing absence

Efficient antibiotic therapy:◦ Decreasing of CRP value◦ Normalizing of CRP concentration means clinical

recovery;

Non – efficient antibiotic therapy:

◦ Persistent increasing of CRP at the end of antibiotic treatment means reappearance of the infectious;

◦ Linear evolution of CRP: - incorrect antibiotic dosages;- the resistance of microorganisms to antibiotics;- forming of a localized suppurate process;- a non – infectious disease;- severe prognosis.

The efficiency of antimicrobial therapy

Blood sampling through catheters; Using just one single bottle instead of three; Cultivation of some quantity of blood in the same

bottles used for other analysis (CRP, glucose, calcium).

Wrong techniques:

Woman, 62 old years, is admitted to hospital with echocardiography diagnosis of sub-acute endocarditis.

From 3 blood cultures was isolated Enterococcus spp. Difuzimetric antibiogram has the next results:

Ampicillin – Resistant (R)Ceftriaxone – Sensitive (S)Gentamicin (120g) - RClindamycin - SCotrimoxazol - RVancomycin - STeicoplanin - SLinezolid - S

Case 1

Antibiotics: mechanisms of actions

There are 2 types of resistance to beta lactams:

Resistance by producing beta lactamase: this resistance has recently appeared in North America and in Latin America, but none of this kind of strains were detected in Europe.

Resistance by modification of PLP: enterococci are naturally resistant to cephalosporines, oxacilins and monobactams.

(Pyridoxal 5′-phosphate (PLP) is a coenzyme synthesized by all forms of life).

Fenotypes of enterococci resistant to antibiotics

Phenotype VanA VanB VanD VanE

MIC vancomycin (ml/l) 60 - > 1024 4 - 1000 64 16

MIC teicoplanin (mg/l) 16 - 512 0.5 - 1 4 0.5

Expression inductible inductible constitutive inductible

Genetic support Seldom plasmidicTransposon Tn1546

Seldom chromosomal chromosomal Chromosomal

Transferable by conjugation

+ + - _

Bacterial species E. faeeciumE faecalis, E. avium,

E. gallinarium, E. flavescens

E. faeciumE. faecalis

E. faecium E. faecalis

The acquired resistance phenotypes of enterococci to glycopeptides

Interpretative antibiogram

• The resistance to Ampicillin and the high level of resistance for Gentamicin compromise the synergic effect of this association;

• This phenotype of resistance is frequent for E. faecium;

• Sensibility testing for Ceftriaxone is incorrect and the reported result is false, because is known that the enterococci are naturally resistant to cephalosporins;

• The sensitivity for clindamycin and cotrimoxazole is also false; this can be explained because of the species identification;

Patient, 68 years old, diabetes, AVC, comma, has received medication through catheter. After 2 weeks present fever and inflammatory syndrome at the place of catheter insertion.

Blood culture made through a non catheterized vein was positive for Klebsiella pneumoniae with the next sensitivity to antibiotics:

Amoxicillin - R Gentamicin - R Amoxicillin +Clavulanic acid - R Amikacin - S

Ticarcillin - R Ciprofloxacin - R Piperacilin + Tazobactam - I Cotrimoxazole - R Ceftazidim - R (sinergy with clavulanic acid ) Ceftriaxone - S

Imipenem - S

Case 2

The main resistance phenotypes of E. coli & K. pneumoniae

Antibiotic Wild phenotype Low level penicinilase

High level penicinilase

Aminopenicillins S R R

Aminopenicillins +IBL S S I/R

Carboxipenicillins S R R

Ureidipenicillins S I/R I/R

First generation cephalosporins

S I I/R

Second generation cephalosporins

S S S/R

Third generation cephalosporins

S S S

Third generation cephalosporins + IBL

S S S

Cefamicins S S S

Broad spectrum cephalosporins

S S S

carbapenems S S S

Francois Jehl, Monique Chomarat, Michele Weber, Alain Gerard, “De l’antibiogramme a prescription”. Edition bioMerieux, ISBN 973 – 86485-2-1, 2010.

Antibiotic Wild phenotype (low level

penicillinase)

High level penicillinase

ESBL(Extended-

Spectrum Beta-lactamase)

CHN(high level cephalosp.)

Aminopenicillins R R R RAminopenicillins +IBL S R R RCarboxipenicillins R R R RUreidipenicillins I R R RFirst generation cephalosporins

S R R R

Second generation cephalosporins

S I/R R R

Third generation cephalosporins

S S R R

Third generation cephalosporins + IBL

S S S R

Cefamicins S S S RBroad spectrum cephalosporins

S S R S

carbapenems S S S S

The main resistance phenotypes of Klebsiella spp.

Francois Jehl, Monique Chomarat, Michele Weber, Alain Gerard, “De l’antibiogramme a prescription”. Edition bioMerieux, ISBN 973 – 86485-2-1, 2010.

Algorithm for phenotypic detection of ESBLs

EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance, July 2013

• Hospital bacteria which produce beta-lactamase with broad spectrum;

• False sensitivity for Ceftriaxone; this phenotype is known to be resistant for all the Cephalosporins;

• Associated resistance to antibiotics from other category;

• The Carbapenems remain active; Ertapenem is preferred;

• It is compulsory the replacement of the colonized catheter.

Interpretative antibiogram

Patient C.T., 16 old days, premature born, with i. v. catheter soon after birth. Since 2 days present fever 38,1°C, pale skin, cold extremities.

The results of the blood cultures: First blood culture: (time 21.30) was positive for

Staphylococcus aureus, after 18 hours of incubation. Second blood culture (time 23.00) was negative, after 7 days

of incubation.

Antibiogram: Sensitive to: meropeneme, norfloxacine, gentamicin,

vancomycin, cotrimoxazolResistant to: penicillin, oxacillin, erythromycin, tetracycline

Interpret the results and argue. Indicate you treatment option.

Case 3

Mechanism Peniciline G, peniciline A, carboxipeniciline,

ureidopeniciline

Antibiotic + beta lactamase

inhibitor

Peniciline M Cephalosporinecarbapenems

Wild S S S S

Penicilase R S S S

Modifying PLP, mecA gene R R R R

BORSA R S/R R S

MODRSA S S R S

The achieved resistance phenotypes of staphylococci at beta lactams

BORSA: S. aureus borderlineMODSA: modified S. aureus

• Bacteria is resistant to Oxacillin (Meticillin resistant), so it will be resistant to all beta lactam antibiotics, including carbapenems;

• Testing for Vancomycin through difuzimetric antibiogram it is not indicated, because the sensitivity to this antibiotic can be tested trough MIC (minimal inhibitory concentration).

Interpretative antibiogram

Patient D.C., 28 years, was involved in a traffic stroke. He received an arthiculary prosthesis at the knee. 4 days after surgical intervention, the patient present fever, 39,2°C.

Sampling: 3 blood cultures at each 30 minutes.

Results: Blood culture I (time 17.10) and II (time 17.40) was

positive for Staphylococcus epidermidis meticilin-resistent

The third blood culture (time 18.10) was negative after 7 days of incubation.

Interpret and argue the results. Indicate your treatment option.

Case 4

MIC determination:

Select colonies

Prepare inoculumsuspension

Mix well

Standardize inoculum

suspension

Swab plate

Remove sample

Add disks

Incubate overnight

Measure ZonesTransmitted

LightReflected

Light

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Epidemiologic trending and reporting 

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