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‫الجامعة االسالمية‪-‬غزة‪.‬‬
‫كلية علوم الصحة‬
‫قسم العلوم الطبية املخبرية‬
‫وبالتعاون مع‬
‫مؤسسة أطباء بال حدود‪.‬‬
‫ضمن‬
‫مشروع التعلم الخدمي‬
‫(‪)Service learning‬‬
‫اشراف الدكتور‪:‬‬
‫عبد الرؤوف املناعمة‬
(GBS) BACTERIA :GROUP
B STREPTOCOCCI
INTRODUCTION
• GBS Bacteria is the leading infectious
cause of early neonatal morbidity and
mortality in the United States.
• Initial case series reported case-fatality
ratios as high as 50%.
STREPTOCOCCUS AGALACTIA
GROUP B , BETA- HEMOLYTIC STREPTOCOCCI:
• General characteristics :.
• Gram positive
• Hemolysis on Sheep Blood Agar is mostly beta hemolysis.
• Catalase negative
• The group antigen is a cell wall polysaccharide composed
of N_acetylglucoseamine, galactos, and rhamanose.
GBS
Hemolytic
• It hydrolysis hippurate to benzoic
acid and glycine.
• It is resistant to bacitracin antibiotic
disk (and could be differentiated
from S. pyogens which is a beta
_hemolytic and sensitive to this
antibiotic).
STREPTOCOCCUS AGALACTIA:
Habitat:
It is a part of the normal oral and vaginal flora .
Approximately 5-15% of healthy population carry
S. agalactia in the nasopharynx.
It can be found in pharynx and gastrointestinal
tract.
It could be present in a various site in the
newborn and this the most important.
ACQUISITION OF GBS DURING BIRTH
PATHOGENICITY:
1. Puerperal
sepsis.
2. Endocarditis.
3. Neonatal infection (Pneumonia, septicemia and meningitis.
4. Bovine mastitis.
Mortality rate 5-10%,
Inversely proportional to birth weight.
SPECIMEN COLLECTION
• Swabbing both the lower vagina and rectum (through the anal
sphincter) increases the culture yield substantially compared
with sampling the cervix or the vagina without also swabbing
the rectum .
• GBS isolated can remain viable in transport media for several
days at room temperature however, the recovery of isolates
declines during 1–4 days, particularly at high temperatures.
• Even when appropriate transport media are used, the
sensitivity of culture is greatest when the specimen is stored at
4°C before culture and processed within24 hours of collection.
CULTURE OF THE ORGANISM :
• 1. On general media : e.g. :,Blood Agar S. agalactiae produces
larger colonies and more translucent to opaque colonies
surrounded by a zone of beta _hemolysis.
• 2. Selective media(streptococcal Selective Agar(SSA)which
contains the following inhibitory chemicals:
• Crystal violet in low concentration,
• Colistin ,
• Trimethoprim _sulphamethoxazole
• in 5% sheep blood agar.
CATALASE
GBS – catalase negative
HIPPURATE HYDROLYSIS TEST:
• Group B streptococci contain enzyme
hippuricase which can hydrolyze hippuric
acid.
• The products of the hydrolysis of sodium
hippurate are sodium benzoate and
glycine .
• Glycine could be detected by the addition
of ninhydrin which is an oxidizing agent
which also gives a purple color with
glycine.
Purple color is
positive for
hippurate
hydrolysis
CAMP TEST:
• The hemolytic activity of staphylococcal beta lysine on
RBCs increased by an extracellular factor produced by
S.agalactia called the CAMP factor.
• This test is don by making a single streak of
streptococcus on sheep blood agar perpendicular to a
strain of staphylococcus aurous known to produce betalysine .
• The positive result is expressed by a zone of increased
lysis assuming the shape of an arrow head at the junction
of the two streak lines.
CAMP Test
Positive zone
of enhanced
hemolytic
activity (GBS)
BILE ESCULIN:
• This test detect the ability of the organism to grow in the
presence of bile and its ability to hydrolyze esculin and
the production of glucose production of and galactos
esculetin.
• Esculetin reacts with iron salts to for a dark brown or
black complex.
• This test is performed in an appropriate medium
containing bile, esculine, ferric citrate as a source of ferric
ions, and sodium azide to inhibit the growth of gram
negative bacteria.
• This test used to differentiate group D streptococci
(positive), and Streptococcus (negative)
LATEX AGGLUTINATION;
• Antibody coated latex particles serves as the basis for several
commercially available systems for direct detection of
bacterial and other microbial antigens in body fluids .
• Latex agglutination tests are also available to detect
antibodies that develop during certain bacterial infection.
• The advantages of agglutination test is rapidity and its relative
sensitivity.
LATEX AGGLUTINATION TEST:
Positive agglutination
is present
Negative agglutination GBS
GBS is not present
Identification Tests from Enrichment Broth
• Direct testing for GBS from broth can occur
AFTER incubation in enrichment broth
• The following methods are supported for
direct testing of the enrichment broth:
– DNA probe
– Latex agglutination test
– Nucleic acid amplification testing (NAAT)
Antimicrobial
Susceptibility Testing
Antimicrobial
Susceptibility Testing
Standard: Penicillin (PCN) or ampicillin
Alternatives:
• PCN-allergic and low risk for anaphylaxis: cefazolin
• PCN-allergic but high risk for anaphylaxis
depends on susceptibility to clindamycin &
erythromycin
– If susceptible to clindamycin (including lack of
inducible resistance)  clindamycin
– If unknown or not susceptible  vancomycin
Susceptibility of GBS:
• All susceptible to penicillin, ampicillin,
cefotaxime, and vancomycin, however:
– Erythromycin resistance: 46%
– Clindamycin resistance: 24%
Clindamycin & Erythromycin Resistance among
GBS isolates, 2000-2008
Isolates are from CO, GA, MD, MN, NY, and OR. 2007 data excluded
Antimicrobial Susceptibility Testing
• CLSI recommends using either:
– Disk diffusion
– Broth micro dilution
• FDA-cleared/approved commercial system may
also be used
– Testing for inducible clindamycin resistance
• D-zone or other validated test
Antimicrobial Susceptibility: Etest & Disk
Diffusion
Zone of inhibition of
growth for
clindamycin is ≥19
mm (susceptible
Erythromycin MIC
= 0.19µg/ml
Etest
Disk Diffusion
Zone of inhibition of
growth for
erythromycin is ≥21
mm (susceptible
Procedure for D-zone Testing to Detect
Inducible Clindamycin Resistance
•
Use swab to make suspension from 18-24 hr. growth of GBS in
saline or Mueller-Hinton broth
•
Dip sterile swab in adjusted suspension
•
Inoculate entire surface of Mueller-Hinton sheep blood agar
plate
•
Place erythromycin (15µg) disk & clindamycin (2µg) disks 12
mm apart
– Incubate for 20-24 hrs at 35C in 5% CO2
•
Blunting of inhibition zone around clindamycin disk adjacent to
erythromycin disk are considered D-zone positive
– If D-zone positive, report as clindamycin resistant
D-zone Test Result for GBS
Blunting of the inhibition zone
indicating inducible clindamycin
resistance
Antimicrobial Susceptibility Test
Broth Microdilution Dilution
Low
High concentration
MIC 0.06 µg/ml
Penicillin
MIC 8 µg/ml
Erythromycin
Sterile control
(MIC) Minimum Inhibitory Concentration
Growth Control
Other changes: Bacteriuria
• 2002 guidelines required labs to report ANY quantity of
GBS found in urine cultures
• Required great deal of lab time
• Studies of bacteriuria as evidence of ‘heavy’
colonization have used ≥104 CFU/mL as cutoff
• Difficult with available data to determine significance of
lower colony counts
• 2010 recommendation is to report positive urine
cultures with ≥104 cfu/mL of GBS
ANTIBIOTICS:





Note that antenatal antibiotics not shown to have benefit for
vaginal colonisation with Group B streptococci.
1.2 g IV Benzyl penicillin initially and then 0.6g
4 hrly during labour.
If penicillin allergic (and no history of
anaphylaxis), cephazolin 2g initially then 1g
8hrly
If prior anaphylactic reaction to penicillin the
either vancomycin 1g 12 hrly, or clindamycin
600mg 8hrly are indicated.
(NZ guidelines recommend vancomycin but
clindamycin is fine if Gp B strep isolated and
known to be susceptible.)


This will decrease incidence of invasive disease in
new-borns by approximately 90%, most effective
if antibiotic started more than 4 hrs before
delivery.
If maternal chorioamnionitis (intra-partum fever
with two of the following signs: foetal tachycardia,
uterine tenderness, offensive vaginal discharge or
increased maternal WCC) then penicillin alone is
insufficient. Broad spectrum abx required eg
Amoxycillin clavulanate
RECOMMENDATIONS
RECOMMENDATIONS
• Pregnant women should undergo vaginal-rectal screening for
GBS colonization at 35-37 weeks.
• Intrapartum antibiotic prophylaxis (IAP) is recommended for:
• o Women who delivered a previous infant with GBS disease
• o Women with GBS bacteriuria in the current pregnancy
• o Women with a GBS-positive screening result in the current
pregnancy o Women with unknown GBS status who deliver at
less than 37 weeks’ gestation,
• have an intrapartum temperature of 100.4F or greater,
• or have rupture of membranes for 18 hours or longer. •
• Penicillin remains the preferred agent with ampicillin an
acceptable alternative .
RECOMMENDATIONS
• personal hygiene.
• Doctors and nurses are recommended to wash your hands
before and after each screening process.
• Sterilization of instruments used in the process of birth after
each case.
• The need to wear gloves at every screening process for
pregnant women by doctors and nurses.
• Change beds linens after each case in order to avoid the
transfer of the microbe.
RECOMMENDATIONS
• Laboratory methods for the isolation, identification or detection of
GBS should be made available at the Ministry of Health Labs
• Updated algorithms for GBS screening and intrapartum
chemoprophylaxis for women with preterm labor or preterm
premature rupture of membranes,
• A change in the recommended dose of penicillin-G for
chemoprophylaxis,
• Updated prophylaxis regimens for women with penicillin allergy.
• A revised algorithm for management of newborns with respect to
risk for early-onset GBS disease .
MAIN MESSAGES
• GBS infection/colonization is an essential part of mother/child
care.
• Every pregnant women should be examined for the presence of
this microbe
• Vaginal and rectal swabs are preferred for the lab detection
• Several methods are available for the detection including
culture, serology, and NAAT
• Intrapartum antibiotic administration proved effective in
preventing diseases for both mother and neonates
‫نشكر لكم حضوركم وحسن استماعكم‬
‫• مع حتيات‪:‬‬
‫• خديجة طالل الحلو‬
‫• ياسمين جالل الدنف‪.‬‬
‫•‬
‫حنين التلبانى‪.‬‬
‫• أريج راشد‬
‫الدريملي‪.‬‬
‫قسم العلوم الطبية املخبرية‬